Polypeptides having trehalase activity and the use thereof in process of producing fermentation products

ABSTRACT

The present invention relates to polypeptides having trehalase activity. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using a trehalase of the invention, in particular a process of producing a fermentation product, such as ethanol.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national application ofPCT/US2016/037224 filed Jun. 13, 2016, which claims priority or thebenefit under 35 U.S.C. 119 of U.S. application No. 62/181,538 filedJun. 18, 2015, the contents of which are fully incorporated herein byreference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to polypeptides having treahalase activityand polynucleotides encoding the polypeptides. The invention alsorelates to nucleic acid constructs, vectors, and host cells comprisingthe polynucleotides as well as methods of producing the polypeptides.The invention also relates to processes of producing fermentationproducts using a trehalase of the invention.

Description of the Related Art

Trehalose is a stable disaccharide sugar consisting of two sugarmonomers (glucose). Trehalose is accumulated in yeast as a response tostress in up to 10-15% of cell dry weight (GrBa et al. (1975) Eur. J.Appl. Microbiol. 2:29-37). Trehalose cannot be metabolized by the yeast.The enzyme trehalase cleaves trehalose into two glucose units.

Trehalases are classified in EC 3.2.1.28 (alpha,alpha-trehalase) and EC.3.2.1.93 (alpha,alpha-phosphotrehalase). The EC classes are based onrecommendations of the Nomenclature Committee of the International Unionof Biochemistry and Molecular Biology (IUBMB). Description of EC classescan be found on the internet. The two enzyme classes are both referredto as “trehalases”. Examples of neutral trehalases include treahalasesfrom Saccharomyces cerevisiae (Londesborouh et al. (1984)Characterization of two trehalases from baker's yeast” Biochem J 219,511-518; Mucor roxii (Dewerchin et al (1984), “Trehalase activity andcyclic AMP content during early development of Mucor rouxii spores”, J.Bacteriol. 158, 575-579); Phycomyces blakesleeanus (Thevelein et al(1983), “Glucose-induced trehalase activation and trehalose mobilizationduring early germination of Phycomyces blakesleeanus spores” J. GenMicrobiol. 129, 719-726); Fusarium oxysporium (Amaral et al (1996),“Comparative study of two trehalase activities from Fusarium oxysporiumvar Linii” Can. J Microbiol. 41, 1057-1062). Examples of neutraltrehalases include, but are not limited to, trehalases fromSaccharomyces cerevisiae (Parvaeh et al. (1996) Purification andbiochemical characterization of the ATH1 gene product, vacuolar acidtrehalase from Saccharomyces cerevisae” FEBS Lett. 391, 273-278);Neorospora crassa (Hecker et al (1973), “Location of trehalase in theascospores of Neurospora: Relation to ascospore dormancy andgermination”. J. Bacteriol. 115, 592-599); Chaetomium aureum (Sumida etal. (1989), “Purification and some properties of trehalase fromChaetomium aureum MS-27. J. Ferment. Bioeng. 67, 83-86); Aspergillusnidulans (d'Enfert et al. (1997), “Molecular characterization of theAspergillus nidulans treA gene encoding an acid trehalase required forgrowth on trehalose. Mol. Microbiol. 24, 203-216); Humicola grisea(Zimmermann et al. (1990).” Purification and properties of anextracellular conidial trehalase from Humicola grisea var. thermoidea”,Biochim. Acta 1036, 41-46); Humicola grisea (Cardello et al. (1994), “Acytosolic trehalase from the thermophilhilic fungus Humicola grisea var.thermoidea, Microbiology UK 140, 1671-1677; Scytalidium thermophilum(Kadowaki et al. (1996), “Characterization of the trehalose system fromthe thermophilic fungus Scytalidium thermophilum” Biochim. Biophys. Acta1291, 199-205); and Fusarium oxysporium (Amaral et al (1996),“Comparative study of two trehalase activities from Fusarium oxysporiumvar Linii” Can. J Microbiol. 41, 1057-1062).

A trehalase is also know from soybean (Aeschbachet et al (1999)“Purification of the trehalase GmTRE1 from soybean nodules and cloningof its cDNA”, Plant Physiol 119, 489-496).

Trehalases are also present in small intestine and kidney of mammals.

WO 2009/121058 (Novozymes) concerns a method of fermenting sugarsderived from plant material into a fermentation product, such asethanol, using a fermenting organism by adding one or more trehalaseinto in the fermentation medium.

WO 2012/027374 (Dyadic) discloses a trehalase from Myceliophthorathermophila which can be used in an enzyme mixture for degradinglignocellulosic biomass to fermentable sugars.

WO 2013/148993 (Novozymes) discloses a process of producing afermentation product, such as ethanol, from starch-containing materialby liquefying, saccharifying and fermenting the starch-containingmaterial wherein wherein a carbohydrate-source generating enzyme, acellulolytic composition and a trehalase is present in fermentation. Atrehalase from Trichoderma reesei is disclosed.

WO 2015/065978 (Danisco US Inc.) discloses a method of increasing theproduction of ethanol from a liquefact in a fermentation reactionincluding fermenting the liquefact with a glucoamylase, a fermentingorganism and a trehalase and recovering the ethanol and otherfermentation products at the end of the fermentation.

There is still a need for providing enzymes or enzyme compositionsuitable for use in processes for producing fermentation products, suchas ethanol, in increased yields.

SUMMARY OF THE INVENTION

The present invention relates to polypeptides having treahalase activityand polynucleotides encoding the polypeptides. The invention alsorelates to processes of producing fermentation products using atrehalase of the invention. The trehalases concerned are E.C. 3.2.1.28and further belong to Family 37 Glucoside Hydrolases (“GH37”) as definedby CAZY (available on the world wide web).

The inventors have found, purified and characterized two trehalases(shown in SEQ ID NO: 30 herein and 4 herein, respectively) having highthermostability and a broad pH range. It was also found that anincreased ethanol yield can be obtained when adding a trehalase of theinvention in fermentation in a process of the invention.

A trehalase of the invention can be used in any yeast fermentationproduct production process, in particular ethanol production process.The treahalase can be used as an exogenous enzyme or can be expressed ina fermentation product producing organism, in particular a yeast strainproducing ethanol, especially a strain of Sacchariomyces, in particulara Saccharomyces cerevisiae strain.

Accordingly, in the first aspect the present invention relates topolypeptides having trehalase activity selected from the groupconsisting of:

-   -   (a) a polypeptide having at least 90% sequence identity to the        mature polypeptide of SEQ ID NO: 30 or SEQ ID NO: 2 or at least        80% sequence identity to the mature polypeptide of SEQ ID NO: 4;    -   (b) a polypeptide encoded by a polynucleotide that hybridizes        under very high stringency conditions with (i) the mature        polypeptide coding sequence of SEQ ID NO: 29, SEQ ID NO: 1 or        SEQ ID NO: 3, (ii) the cDNA sequence thereof, or (iii) the        full-length complement of (i) or (ii);    -   (c) a polypeptide encoded by a polynucleotide having at least        90% sequence identity to the mature polypeptide coding sequence        of SEQ ID NO: 29 or SEQ ID NO: 1 or at least 80% sequence        identity to the mature polypeptide coding sequence of SEQ ID NO:        3 or the cDNA sequence thereof;    -   (d) a variant of the mature polypeptide of SEQ ID NO: 30, SEQ ID        NO: 2 or SEQ ID NO: 4 comprising a substitution, deletion,        and/or insertion at one or more (e.g., several) positions; and    -   (e) a fragment of the polypeptide of (a), (b), (c), or (d) that        has trehalase activity.

The mature polypeptide of SEQ ID NO: 30 is shown as amino acids 21-694.The mature polypeptide of SEQ ID NO: 2 is shown as amino acids 21-697.The mature polypeptide of SEQ ID NO: 4 is shown as amino acids 21-690.The signal of SEQ ID NO: 30 is shown as amino acids 1-20. The signal ofSEQ ID NO: 2 is shown as amino acids 1-20. The signal of SEQ ID NO: 4 isshown as amino acids 21-690.

The present invention also relates to polynucleotides encoding thepolypeptides having trehalase activity of the present invention; nucleicacid constructs; recombinant expression vectors; recombinant host cellscomprising the polynucleotides; and methods of producing thepolypeptides.

In an aspect the present invention also relates to processes ofproducing a fermentation product, in particular ethanol, comprising

-   -   (a) liquefying a starch-containing material with an        alpha-amylase;    -   optionally pre-saccharifying the liquefied material before step        (b);    -   (b) saccharifying the liquefied material;    -   (c) fermenting using a fermentation organism;    -   wherein        -   i) a glucoamylase;        -   ii) a trehalase of the invention;        -   iii) optionally a cellulolytic enzyme composition and/or a            protease;            are present and/or added during    -   saccharification step (b);    -   fermentation step (c);    -   simultaneous saccharification and fermentation;    -   optionally the presaccharification step before step (b).

Liquefaction in step (a) may be carried out at a temperature above theinitial gelatinization temperature, in particular between 80° C.-90° C.

In an embodiment the invention relates to processes of producingfermentation products from starch-containing material comprising:

-   -   (i) saccharifying a starch-containing material at a temperature        below the initial gelatinization temperature; and    -   (ii) fermenting using a fermentation organism;    -   wherein saccharification and/or fermentation is done in the        presence of the following enzymes: glucoamylase, alpha-amylase,        trehalase of the invention, and optionally a cellulolytic enzyme        composition and/or a protease.

In an embodiment the invention relates to processes of producing afermentation product from pretreated cellulosic material, comprising:

-   -   (a) hydrolyzing said pretreated cellulosic material with a        cellulolytic enzyme composition;    -   (b) fermenting using a fermenting organism; and    -   (c) optionally recovering the fermentation product;    -   wherein a trehalase of the invention is added and/or present in        hydrolysis step (a) and/or fermentation step (b).

In an embodiment the trehalase may be added to the thin stillage, i.e.,at the backend of a starch based or a cellulosic material based process.

A trehalase of the invention may be present and/or added insaccharification/hydrolysis and/or fermentation in a fermentationproduct producing process of the invention in any suitable amount. In apreferred embodiment the trehalase is present and/or added in an amountbetween 0.01-20 ug EP trehalase/g DS, such as between 0.05-15 ug EPterhalase/g DS, such as between 0.5 and 10 ug EP trehalase/g DS.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the glucose production by trehalase addition in supernatantof ferm drop.

FIG. 2 shows the glucose production by Ms trehalase (SEQ ID NO: 30) andGH65 Tr trehalase (SEQ ID NO: 31) addition in supernatant of ferm drop.

DEFINITIONS

Trehalase: The term “trehalase” means an enzyme which degrades trehaloseinto its unit monosaccharides (i.e., glucose). Trehalases are classifiedin EC 3.2.1.28 (alpha,alpha-trehalase) and EC. 3.2.1.93(alpha,alpha-phosphotrehalase). The EC classes are based onrecommendations of the Nomenclature Committee of the International Unionof Biochemistry and Molecular Biology (IUBMB). Description of EC classescan be found on the internet. Trehalases are enzymes that catalyze thefollowing reactions:

EC 3.2.1.28:Alpha,alpha-trehalose+H₂O⇔2 D-glucose;EC 3.2.1. 93:Alpha,alpha-trehalose 6-phosphate+H₂O⇔D-glucose+D-glucose 6-phosphate.

For purposes of the present invention, trehalase activity may bedetermined according to “Trehalase Assay” procedure described in the“Materials & Methods”-section. In one aspect, the polypeptides of thepresent invention have at least 20%, e.g., at least 40%, at least 50%,at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, orat least 100% of the trehalase activity of the mature polypeptide of SEQID NO: 30, SEQ ID NO: 2 or SEQ ID NO: 4, respectively. In a preferredembodiment a trehalase of the invention is a Family 37 GlycosideHydrolase (“GH37 trehalase”).

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Catalytic domain: The term “catalytic domain” means the region of anenzyme containing the catalytic machinery of the enzyme.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a polypeptide. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG, or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding a maturepolypeptide of the present invention. Each control sequence may benative (i.e., from the same gene) or foreign (i.e., from a differentgene) to the polynucleotide encoding the polypeptide or native orforeign to each other. Such control sequences include, but are notlimited to, a leader, polyadenylation sequence, propeptide sequence,promoter, signal peptide sequence, and transcription terminator. At aminimum, the control sequences include a promoter, and transcriptionaland translational stop signals. The control sequences may be providedwith linkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe polynucleotide encoding a polypeptide.

Expression: The term “expression” includes any step involved in theproduction of a polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to control sequences that provide forits expression.

Fragment: The term “fragment” means a polypeptide having one or more(e.g., several) amino acids absent from the amino and/or carboxylterminus of a mature polypeptide or domain; wherein the fragment hastrehalase activity. In one aspect, a fragment contains at least 592amino acid residues, at least 627 amino acid residues, or at least 662amino acid residues of SEQ ID NO: 2. In another aspect, a fragmentcontains at least 587 amino acid residues, at least 621 amino acidresidues, or at least 655 amino acid residues of SEQ ID NO: 4.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Isolated: The term “isolated” means a substance in a form or environmentthat does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., recombinantproduction in a host cell; multiple copies of a gene encoding thesubstance; and use of a stronger promoter than the promoter naturallyassociated with the gene encoding the substance). An isolated substancemay be present in a fermentation broth sample; e.g. a host cell may begenetically modified to express the polypeptide of the invention. Thefermentation broth from that host cell will comprise the isolatedpolypeptide.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 21 to 694 of SEQ ID NO: 30. Amino acids 1 to20 of SEQ ID NO: 30 are the signal peptide. In one aspect, the maturepolypeptide is amino acids 21 to 697 of SEQ ID NO: 2. Amino acids 1 to20 of SEQ ID NO: 2 are a signal peptide. In another aspect, the maturepolypeptide is amino acids 21 to 690 of SEQ ID NO: 4. Amino acids 1 to20 of SEQ ID NO: 4 are a signal peptide. The signal peptides aredetermined using the SignalP program (Nielsen et al., 1997, ProteinEngineering 10: 1-6). It is known in the art that a host cell mayproduce a mixture of two of more different mature polypeptides (i.e.,with a different C-terminal and/or N-terminal amino acid) expressed bythe same polynucleotide. It is also known in the art that different hostcells process polypeptides differently, and thus, one host cellexpressing a polynucleotide may produce a different mature polypeptide(e.g., having a different C-terminal and/or N-terminal amino acid) ascompared to another host cell expressing the same polynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving trehalase activity.

In one aspect, the mature polypeptide coding sequence is nucleotides(101) . . . (2326) of SEQ ID NO: 29 or the cDNA sequence thereof shownas nucleotides 501-679, 366-1025, 1084-2326.

In one aspect, the mature polypeptide coding sequence is nucleotides(501) . . . (2814) of SEQ ID NO: 1 or the cDNA sequence thereof shown asnucleotides 501-679, 766-1425, 1484-2676 and 2756-2814. Nucleotides 501to 560 of SEQ ID NO: 1 encode a signal peptide. In another aspect, themature polypeptide coding sequence is nucleotides 501-2701 of SEQ ID NO:3 or the cDNA sequence thereof shown as nucleotides 501-679, 752-1411,and 1471-2701. Nucleotides 501 to 560 of SEQ ID NO: 3 encode a signalpeptide.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the—nobrief option) is usedas the percent identity and is calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

Stringency conditions: The term “high stringency conditions” means forprobes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at65° C.

The term “very high stringency conditions” means for probes of at least100 nucleotides in length, prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 50% formamide, following standard Southern blottingprocedures for 12 to 24 hours. The carrier material is finally washedthree times each for 15 minutes using 2×SSC, 0.2% SDS at 70° C.]

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides absent from the 5′ and/or 3′ end of amature polypeptide coding sequence; wherein the subsequence encodes afragment having trehalase activity. In one aspect, a subsequencecontains at least 1776 nucleotides, at least 1881 nucleotides, or atleast 1962 nucleotides of SEQ ID NO: 29 or SEQ ID NO: 1. In one aspect,a subsequence contains at least 1761 nucleotides, at least 1863nucleotides, or at least 1965 nucleotides of SEQ ID NO: 3.

Variant: The term “variant” means a polypeptide having trehalaseactivity comprising an alteration, i.e., a substitution, insertion,and/or deletion, at one or more (e.g., several) positions. Asubstitution means replacement of the amino acid occupying a positionwith a different amino acid; a deletion means removal of the amino acidoccupying a position; and an insertion means adding an amino acidadjacent to and immediately following the amino acid occupying aposition.

DETAILED DESCRIPTION OF THE INVENTION

Polypeptides Having Trehalase Activity

In an embodiment, the present invention relates to polypeptides having asequence identity to the mature polypeptide of SEQ ID NO: 30 of at least80, at least 85, at least 90%, at least 91%, at least 92%, at least 93%,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%, which have trehalase activity. In one aspect, thepolypeptides differ by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, or 10, from the mature polypeptide of SEQ ID NO: 30.

In an embodiment, the present invention relates to polypeptides having asequence identity to the mature polypeptide of SEQ ID NO: 2 of at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100%, which have trehalase activity. In oneaspect, the polypeptides differ by up to 10 amino acids, e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID NO: 2.

In a particular embodiment the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 30 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 70% of the trehalase activity of the mature polypeptideof SEQ ID NO: 30.

In a particular embodiment the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 2 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 70% of the trehalase activity of the mature polypeptideofSEQ ID NO: 2.

In a particular embodiment the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 2 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast 75% of the trehalase activity of the mature polypeptide of SEQ IDNO: 30 or SEQ ID NO: 2.

In a particular embodiment the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 30 or SEQ IDNO: 2 of at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%, and wherein thepolypeptide has at least at least 80% of the trehalase activity of themature polypeptide of SEQ ID NO: 30 or SEQ ID NO: 2.

In a particular embodiment the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 30 or SEQ IDNO: 2 of at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%, and wherein thepolypeptide has at least at least 85% of the trehalase activity of themature polypeptide of SEQ ID NO: 30 or SEQ ID NO: 2.

In a particular embodiment the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 30 or SEQ IDNO: 2 of at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%, and wherein thepolypeptide has at least at least 90% of the trehalase activity of themature polypeptide of SEQ ID NO: 30 or SEQ ID NO: 2.

In a particular embodiment the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 30 or SEQ IDNO: 2 of at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%, and wherein thepolypeptide has at least 95% of the trehalase activity of the maturepolypeptide of SEQ ID NO: 30 or SEQ ID NO: 2.

In a particular embodiment the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 30 or SEQ IDNO: 2 of at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%, and wherein thepolypeptide has at least 100% of the trehalase activity of the maturepolypeptide of SEQ ID NO: 30 or SEQ ID NO: 2.

In an embodiment, the present invention relates to polypeptides having asequence identity to the mature polypeptide of SEQ ID NO: 4 of at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100%, which have trehalase activity. In oneaspect, the polypeptides differ by up to 10 amino acids, e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID NO: 4.

In a particular embodiment the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 4 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 70% of the trehalase activity of the mature polypeptideof SEQ ID NO: 4.

In a particular embodiment the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 4 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 75% of the trehalase activity of the mature polypeptideof SEQ ID NO: 4.

In a particular embodiment the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 4 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 80% of the trehalase activity of the mature polypeptideof SEQ ID NO: 4.

In a particular embodiment the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 4 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 85% of the trehalase activity of the mature polypeptideof SEQ ID NO: 4.

In a particular embodiment the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 4 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 90% of the trehalase activity of the mature polypeptideof SEQ ID NO: 4.

In a particular embodiment the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 4 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast 95% of the trehalase activity of the mature polypeptide of SEQ IDNO: 4.

In a particular embodiment the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 4 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast 100% of the trehalase activity of the mature polypeptide of SEQ IDNO: 4.

In an embodiment, the polypeptide has been isolated. A polypeptide ofthe present invention preferably comprises or consists of the amino acidsequence of SEQ ID NO: 30 or SEQ ID NO: 2 or an allelic variant thereof;or is a fragment thereof having trehalase activity. In another aspect,the polypeptide comprises or consists of the mature polypeptide of SEQID NO: 30 or SEQ ID NO: 2. In another aspect, the polypeptide comprisesor consists of amino acids 21 to 694 of SEQ ID NO: 30 or amino acids 21to 697 of SEQ ID NO: 2.

In an embodiment, the polypeptide has been isolated. A polypeptide ofthe present invention preferably comprises or consists of the amino acidsequence of SEQ ID NO: 4 or an allelic variant thereof; or is a fragmentthereof having trehalase activity. In another aspect, the polypeptidecomprises or consists of the mature polypeptide of SEQ ID NO: 4. Inanother aspect, the polypeptide comprises or consists of amino acids 21to 690 of SEQ ID NO: 4.

In another embodiment, the present invention relates to a polypeptidehaving trehalase activity encoded by a polynucleotide that hybridizesunder high stringency conditions or very high stringency conditions with(i) the mature polypeptide coding sequence of SEQ ID NO: 29 or SEQ IDNO: 1, (ii) the cDNA sequence thereof, or (iii) the full-lengthcomplement of (i) or (ii) (Sambrook et al., 1989, Molecular Cloning, ALaboratory Manual, 2d edition, Cold Spring Harbor, N.Y.). In anembodiment, the polypeptide has been isolated.

In another embodiment, the present invention relates to a polypeptidehaving trehalase activity encoded by a polynucleotide that hybridizesunder high stringency conditions or very high stringency conditions with(i) the mature polypeptide coding sequence of SEQ ID NO: 3, (ii) thecDNA sequence thereof, or (iii) the full-length complement of (i) or(ii) (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2dedition, Cold Spring Harbor, N.Y.). In an embodiment, the polypeptidehas been isolated.

The polynucleotide of SEQ ID NO: 29 or SEQ ID NO: 1 or a subsequencethereof, as well as the polypeptide of SEQ ID NO: 30 or SEQ ID NO: 2 ora fragment thereof may be used to design nucleic acid probes to identifyand clone DNA encoding polypeptides having trehalase activity fromstrains of different genera or species according to methods well knownin the art. In particular, such probes can be used for hybridizationwith the genomic DNA or cDNA of a cell of interest, following standardSouthern blotting procedures, in order to identify and isolate thecorresponding gene therein. Such probes can be considerably shorter thanthe entire sequence, but should be at least 15, e.g., at least 25, atleast 35, or at least 70 nucleotides in length. Preferably, the nucleicacid probe is at least 100 nucleotides in length, e.g., at least 200nucleotides, at least 300 nucleotides, at least 400 nucleotides, atleast 500 nucleotides, at least 600 nucleotides, at least 700nucleotides, at least 800 nucleotides, or at least 900 nucleotides inlength. Both DNA and RNA probes can be used. The probes are typicallylabeled for detecting the corresponding gene (for example, with ³²P, ³H,³⁵S, biotin, or avidin). Such probes are encompassed by the presentinvention.

The polynucleotide of SEQ ID NO: 3 or a subsequence thereof, as well asthe polypeptide of SEQ ID NO: 4 or a fragment thereof may be used todesign nucleic acid probes to identify and clone DNA encodingpolypeptides having trehalase activity from strains of different generaor species according to methods well known in the art. In particular,such probes can be used for hybridization with the genomic DNA or cDNAof a cell of interest, following standard Southern blotting procedures,in order to identify and isolate the corresponding gene therein. Suchprobes can be considerably shorter than the entire sequence, but shouldbe at least 15, e.g., at least 25, at least 35, or at least 70nucleotides in length. Preferably, the nucleic acid probe is at least100 nucleotides in length, e.g., at least 200 nucleotides, at least 300nucleotides, at least 400 nucleotides, at least 500 nucleotides, atleast 600 nucleotides, at least 700 nucleotides, at least 800nucleotides, or at least 900 nucleotides in length. Both DNA and RNAprobes can be used. The probes are typically labeled for detecting thecorresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin).Such probes are encompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a polypeptide having trehalase activity. Genomic or other DNAfrom such other strains may be separated by agarose or polyacrylamidegel electrophoresis, or other separation techniques. DNA from thelibraries or the separated DNA may be transferred to and immobilized onnitrocellulose or other suitable carrier material. In order to identifya clone or DNA that hybridizes with SEQ ID NO: 1 or a subsequencethereof or SEQ ID NO: 3 or a subsequence thereof, the carrier materialis used in a Southern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto (i) SEQ ID NO: 29 or SEQ ID NO: 1; (ii) the mature polypeptide codingsequence of SEQ ID NO: 29 or SEQ ID NO: 1; (iii) the cDNA sequencethereof; (iv) the full-length complement thereof; or (v) a subsequencethereof; under high to very high stringency conditions.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto (i) SEQ ID NO: 3; (ii) the mature polypeptide coding sequence of SEQID NO: 3; (iii) the cDNA sequence thereof; (iv) the full-lengthcomplement thereof; or (v) a subsequence thereof; under high to veryhigh stringency conditions.

Molecules to which the nucleic acid probe hybridizes under theseconditions can be detected using, for example, X-ray film or any otherdetection means known in the art.

In another embodiment, the present invention relates to a polypeptidehaving trehalase activity encoded by a polynucleotide having a sequenceidentity to the mature polypeptide coding sequence of SEQ ID NO: 29 orthe cDNA sequence thereof of at least 60%, e.g., at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100%. In a furtherembodiment, the polypeptide has been isolated.

In another embodiment, the present invention relates to a polypeptidehaving trehalase activity encoded by a polynucleotide having a sequenceidentity to the mature polypeptide coding sequence of SEQ ID NO: 1 orthe cDNA sequence thereof of at least 60%, e.g., at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100%. In a furtherembodiment, the polypeptide has been isolated.

In another embodiment, the present invention relates to a polypeptidehaving trehalase activity encoded by a polynucleotide having a sequenceidentity to the mature polypeptide coding sequence of SEQ ID NO: 3 orthe cDNA sequence thereof of at least 60%, e.g., at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100%. In a furtherembodiment, the polypeptide has been isolated.

In another embodiment, the present invention relates to variants of themature polypeptide of SEQ ID NO: 30 comprising a substitution, deletion,and/or insertion at one or more (e.g., several) positions. In anembodiment, the number of amino acid substitutions, deletions and/orinsertions introduced into the mature polypeptide of SEQ ID NO: 30 is upto 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In another embodiment, the present invention relates to variants of themature polypeptide of SEQ ID NO: 2 comprising a substitution, deletion,and/or insertion at one or more (e.g., several) positions. In anembodiment, the number of amino acid substitutions, deletions and/orinsertions introduced into the mature polypeptide of SEQ ID NO: 2 is upto 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In another embodiment, the present invention relates to variants of themature polypeptide of SEQ ID NO: 4 comprising a substitution, deletion,and/or insertion at one or more (e.g., several) positions. In anembodiment, the number of amino acid substitutions, deletions and/orinsertions introduced into the mature polypeptide of SEQ ID NO: 4 is upto 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

The amino acid changes may be of a minor nature, that is conservativeamino acid substitutions or insertions that do not significantly affectthe folding and/or activity of the protein; small deletions, typicallyof 1-30 amino acids; small amino- or carboxyl-terminal extensions, suchas an amino-terminal methionine residue; a small linker peptide of up to20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant moleculesare tested for trehalase activity to identify amino acid residues thatare critical to the activity of the molecule. See also, Hilton et al.,1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzyme orother biological interaction can also be determined by physical analysisof structure, as determined by such techniques as nuclear magneticresonance, crystallography, electron diffraction, or photoaffinitylabeling, in conjunction with mutation of putative contact site aminoacids. See, for example, de Vos et al., 1992, Science 255: 306-312;Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992,FEBS Lett. 309: 59-64. The identity of essential amino acids can also beinferred from an alignment with a related polypeptide.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

The polypeptide may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The polypeptide may be a fusion polypeptide or cleavable fusionpolypeptide in which another polypeptide is fused at the N-terminus orthe C-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

Sources of Polypeptides Having Trehalase Activity

A polypeptide having trehalase activity of the present invention may beobtained from microorganisms of any genus. For purposes of the presentinvention, the term “obtained from” as used herein in connection with agiven source shall mean that the polypeptide encoded by a polynucleotideis produced by the source or by a strain in which the polynucleotidefrom the source has been inserted. In one aspect, the polypeptideobtained from a given source is secreted extracellularly.

In another aspect, the polypeptide is a Myceliophthora sepedoniumpolypeptide having treahalase or a Chaetomium virescens polypeptidehaving trehalase activity.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The polypeptide may be identified and obtained from other sourcesincluding microorganisms isolated from nature (e.g., soil, composts,water, etc.) or DNA samples obtained directly from natural materials(e.g., soil, composts, water, etc.) using the above-mentioned probes.Techniques for isolating microorganisms and DNA directly from naturalhabitats are well known in the art. A polynucleotide encoding thepolypeptide may then be obtained by similarly screening a genomic DNA orcDNA library of another microorganism or mixed DNA sample. Once apolynucleotide encoding a polypeptide has been detected with theprobe(s), the polynucleotide can be isolated or cloned by utilizingtechniques that are known to those of ordinary skill in the art (see,e.g., Sambrook et al., 1989, supra).

Polynucleotides

The present invention also relates to polynucleotides encoding apolypeptide of the present invention, as described herein. In anembodiment, the polynucleotide encoding the polypeptide of the presentinvention has been isolated.

The techniques used to isolate or clone a polynucleotide are known inthe art and include isolation from genomic DNA or cDNA, or a combinationthereof. The cloning of the polynucleotides from genomic DNA can beeffected, e.g., by using the well-known polymerase chain reaction (PCR)or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligation activated transcription (LAT) andpolynucleotide-based amplification (NASBA) may be used. Thepolynucleotides may be cloned from a strain of Myceliophthora, or arelated organism or Chaetomium, or a related organism and thus, forexample, may be an allelic or species variant of the polypeptideencoding region of the polynucleotide.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the expression of the coding sequence in asuitable host cell under conditions compatible with the controlsequences.

The polynucleotide may be manipulated in a variety of ways to providefor expression of the polypeptide. Manipulation of the polynucleotideprior to its insertion into a vector may be desirable or necessarydepending on the expression vector. The techniques for modifyingpolynucleotides utilizing recombinant DNA methods are well known in theart.

The control sequence may be a promoter, a polynucleotide that isrecognized by a host cell for expression of a polynucleotide encoding apolypeptide of the present invention. The promoter containstranscriptional control sequences that mediate the expression of thepolypeptide. The promoter may be any polynucleotide that showstranscriptional activity in the host cell including variant, truncated,and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes,Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Dania (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase III,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor, as well as the NA2-tpi promoter (a modified promoterfrom an Aspergillus neutral alpha-amylase gene in which the untranslatedleader has been replaced by an untranslated leader from an Aspergillustriose phosphate isomerase gene; non-limiting examples include modifiedpromoters from an Aspergillus niger neutral alpha-amylase gene in whichthe untranslated leader has been replaced by an untranslated leader froman Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerasegene); and variant, truncated, and hybrid promoters thereof. Otherpromoters are described in U.S. Pat. No. 6,011,147.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminator isoperably linked to the 3′-terminus of the polynucleotide encoding thepolypeptide. Any terminator that is functional in the host cell may beused in the present invention.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans acetamidase, Aspergillusnidulans anthranilate synthase, Aspergillus niger glucoamylase,Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase,Fusarium oxysporum trypsin-like protease, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase III,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leader isoperably linked to the 5′-terminus of the polynucleotide encoding thepolypeptide. Any leader that is functional in the host cell may be used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a polypeptide anddirects the polypeptide into the cell's secretory pathway. The 5′-end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′-end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. A foreign signal peptide coding sequence may be required wherethe coding sequence does not naturally contain a signal peptide codingsequence. Alternatively, a foreign signal peptide coding sequence maysimply replace the natural signal peptide coding sequence in order toenhance secretion of the polypeptide. However, any signal peptide codingsequence that directs the expressed polypeptide into the secretorypathway of a host cell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of apolypeptide and the signal peptide sequence is positioned next to theN-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the polypeptide relative to the growth of the host cell.Examples of regulatory sequences are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysequences in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter,and Trichoderma reesei cellobiohydrolase II promoter may be used. Otherexamples of regulatory sequences are those that allow for geneamplification. In eukaryotic systems, these regulatory sequences includethe dihydrofolate reductase gene that is amplified in the presence ofmethotrexate, and the metallothionein genes that are amplified withheavy metals. In these cases, the polynucleotide encoding thepolypeptide would be operably linked to the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleotideand control sequences may be joined together to produce a recombinantexpression vector that may include one or more convenient restrictionsites to allow for insertion or substitution of the polynucleotideencoding the polypeptide at such sites. Alternatively, thepolynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin, or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, adeA (phosphoribosylaminoimidazole-succinocarboxam ide synthase), adeB(phosphoribosyl-aminoimidazole synthase), amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene. Preferred for use in a Trichodermacell are adeA, adeB, amdS, hph, and pyrG genes.

The selectable marker may be a dual selectable marker system asdescribed in WO 2010/039889. In one aspect, the dual selectable markeris an hph-tk dual selectable marker system.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMß1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a polypeptide. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the production of a polypeptide of thepresent invention. A construct or vector comprising a polynucleotide isintroduced into a host cell so that the construct or vector ismaintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thepolypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell including, butnot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397) or conjugation (see, e.g.,Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804), or conjugation(see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, anymethod known in the art for introducing DNA into a host cell can beused.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a polypeptideof the present invention, comprising (a) cultivating a cell, which inits wild-type form produces the polypeptide, under conditions conducivefor production of the polypeptide; and optionally, (b) recovering thepolypeptide. In one aspect, the cell is a Myceliophthora cell. Inanother aspect, the cell is a Myceliophthora sepedonium cell. In oneaspect, the cell is a Chaetomium cell. In another aspect, the cell is aChaetomium virescens cell.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising (a) cultivating a recombinant hostcell of the present invention under conditions conducive for productionof the polypeptide; and optionally, (b) recovering the polypeptide.

The host cells are cultivated in a nutrient medium suitable forproduction of the polypeptide using methods known in the art. Forexample, the cells may be cultivated by shake flask cultivation, orsmall-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptide may be detected using methods known in the art that arespecific for the polypeptides. These detection methods include, but arenot limited to, use of specific antibodies, formation of an enzymeproduct, or disappearance of an enzyme substrate. For example, an enzymeassay may be used to determine the activity of the polypeptide.

The polypeptide may be recovered using methods known in the art. Forexample, the polypeptide may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation. In one aspect, a fermentation broth comprising thepolypeptide is recovered. The polypeptide may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, Janson and Ryden, editors, VCH Publishers, NewYork, 1989) to obtain substantially pure polypeptides.

In an alternative aspect, the polypeptide is not recovered, but rather ahost cell of the present invention expressing the polypeptide is used asa source of the polypeptide.

Production in Plants

The present invention also relates to isolated plants, e.g., atransgenic plant, plant part, or plant cell, comprising a polynucleotideof the present invention so as to express and produce a polypeptide ordomain in recoverable quantities. The polypeptide or domain may berecovered from the plant or plant part. Alternatively, the plant orplant part containing the polypeptide or domain may be used as such forimproving the quality of a food or feed, e.g., improving nutritionalvalue, palatability, and rheological properties, or to destroy anantinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn). Examples of dicot plants are tobacco,legumes, such as lupins, potato, sugar beet, pea, bean and soybean, andcruciferous plants (family Brassicaceae), such as cauliflower, rapeseed, and the closely related model organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.

Plant cells and specific plant cell compartments, such as chloroplasts,apoplasts, mitochondria, vacuoles, peroxisomes and cytoplasm are alsoconsidered to be a plant part.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing the polypeptide or domainmay be constructed in accordance with methods known in the art.

The present invention also relates to methods of producing a polypeptideor domain of the present invention comprising (a) cultivating atransgenic plant or a plant cell comprising a polynucleotide encodingthe polypeptide or domain under conditions conducive for production ofthe polypeptide or domain; and (b) recovering the polypeptide or domain.

Fermentation Broth Formulations or Cell Compositions

The present invention also relates to a fermentation broth formulationor a cell composition comprising a polypeptide of the present invention.The fermentation broth product further comprises additional ingredientsused in the fermentation process, such as, for example, cells(including, the host cells containing the gene encoding the polypeptideof the present invention which are used to produce the polypeptide ofinterest), cell debris, biomass, fermentation media and/or fermentationproducts. In some embodiments, the composition is a cell-killed wholebroth containing organic acid(s), killed cells and/or cell debris, andculture medium.

The term “fermentation broth” as used herein refers to a preparationproduced by cellular fermentation that undergoes no or minimal recoveryand/or purification. For example, fermentation broths are produced whenmicrobial cultures are grown to saturation, incubated undercarbon-limiting conditions to allow protein synthesis (e.g., expressionof enzymes by host cells) and secretion into cell culture medium. Thefermentation broth can contain unfractionated or fractionated contentsof the fermentation materials derived at the end of the fermentation.Typically, the fermentation broth is unfractionated and comprises thespent culture medium and cell debris present after the microbial cells(e.g., filamentous fungal cells) are removed, e.g., by centrifugation.In some embodiments, the fermentation broth contains spent cell culturemedium, extracellular enzymes, and viable and/or nonviable microbialcells.

In an embodiment, the fermentation broth formulation and cellcompositions comprise a first organic acid component comprising at leastone 1-5 carbon organic acid and/or a salt thereof and a second organicacid component comprising at least one 6 or more carbon organic acidand/or a salt thereof. In a specific embodiment, the first organic acidcomponent is acetic acid, formic acid, propionic acid, a salt thereof,or a mixture of two or more of the foregoing and the second organic acidcomponent is benzoic acid, cyclohexanecarboxylic acid, 4-methylvalericacid, phenylacetic acid, a salt thereof, or a mixture of two or more ofthe foregoing.

In one aspect, the composition contains an organic acid(s), andoptionally further contains killed cells and/or cell debris. In oneembodiment, the killed cells and/or cell debris are removed from acell-killed whole broth to provide a composition that is free of thesecomponents.

The fermentation broth formulations or cell compositions may furthercomprise a preservative and/or anti-microbial (e.g., bacteriostatic)agent, including, but not limited to, sorbitol, sodium chloride,potassium sorbate, and others known in the art.

The cell-killed whole broth or composition may contain theunfractionated contents of the fermentation materials derived at the endof the fermentation. Typically, the cell-killed whole broth orcomposition contains the spent culture medium and cell debris presentafter the microbial cells (e.g., filamentous fungal cells) are grown tosaturation, incubated under carbon-limiting conditions to allow proteinsynthesis. In some embodiments, the cell-killed whole broth orcomposition contains the spent cell culture medium, extracellularenzymes, and killed filamentous fungal cells. In some embodiments, themicrobial cells present in the cell-killed whole broth or compositioncan be permeabilized and/or lysed using methods known in the art.

A whole broth or cell composition as described herein is typically aliquid, but may contain insoluble components, such as killed cells, celldebris, culture media components, and/or insoluble enzyme(s). In someembodiments, insoluble components may be removed to provide a clarifiedliquid composition.

The whole broth formulations and cell compositions of the presentinvention may be produced by a method described in WO 90/15861 or WO2010/096673.

Enzyme Compositions

The present invention also relates to compositions comprising apolypeptide having trehalase activity of the present invention.Preferably, the compositions are enriched in such a polypeptide. Theterm “enriched” indicates that the trehalase activity of the compositionhas been increased, e.g., with an enrichment factor of at least 1.1.

The compositions may comprise a polypeptide of the present invention asthe major enzymatic component, e.g., a mono-component composition.Alternatively, the compositions may comprise multiple enzymaticactivities, such as one or more (e.g., several) enzymes selected fromthe group consisting of hydrolase, isomerase, ligase, lyase,oxidoreductase, or transferase, e.g., an alpha-galactosidase,alpha-glucosidase, aminopeptidase, amylase, beta-galactosidase,beta-glucosidase, beta-xylosidase, carbohydrase, carboxypeptidase,catalase, cellobiohydrolase, cellulase, chitinase, cutinase,cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase,esterase, glucoamylase, invertase, laccase, lipase, mannosidase,mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase,polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase,or xylanase.

In an embodiment the composition comprises a trehalase of the inventionand a glucoamylase. In an embodiment the composition comprises atrehalase of the invention and a glucoamylase derived from Talaromycesemersonii (e.g., SEQ ID NO: 13). In an embodiment the compositioncomprises a trehalase of the invention and a glucoamylase derived fromGloeophyllum, such as G. serpiarium (e.g., SEQ ID NO: 19) or G. trabeum(e.g., SEQ ID NO: 20). In an embodiment the composition comprises atrehalase of the invention, a glucoamylase and an alpha-amylase. In anembodiment the composition comprises a trehalase of the invention, aglucoamylase and an alpha-amylase derived from Rhizomucor, preferably astrain the Rhizomucor pusillus, such as a Rhizomucor pusillusalpha-amylase hybrid having an linker (e.g., from Aspergillus niger) andstarch-bonding domain (e.g., from Aspergillus niger). In an embodimentthe composition comprises a trehalase of the invention, a glucoamylase,an alpha-amylase and a cellulolytic enzyme composition. In an embodimentthe composition comprises a trehalase of the invention, a glucoamylase,an alpha-amylase and a cellulolytic enzyme composition, wherein thecellulolytic composition is derived from Trichoderma reesei. In anembodiment the composition comprises a trehalase of the invention, aglucoamylase, an alpha-amylase and a protease. In an embodiment thecomposition comprises a trehalase of the invention, a glucoamylase, analpha-amylase and a protease. The protease may be derived fromThermoascus aurantiacus. In an embodiment the composition comprises atrehalase of the invention, a glucoamylase, an alpha-amylase, acellulolytic enzyme composition and a protease. In an embodiment thecomposition comprises a trehalase of the invention, a glucoamylase,e.g., derived from Talaromyces emersonii, Gloeophyllum serpiarium orGloephyllum trabeum, an alpha-amylase, e.g., derived from Rhizomucorpusillus, in particular one having a linker and starch-binding domain,in particular derived from Aspergillus niger, in particular one havingthe following substitutions: G128D+D143N (using SEQ ID NO: 15 fornumbering); a cellulolytic enzyme composition derived from Trichodermareesei, and a protease, e.g., derived from Thermoascus aurantiacus.

Examples of specifically contemplated secondary enzymes, e.g., aglucoamylase from Talaromyces emersonii shown in SEQ ID NO: 13 herein ora glucoamylase having, e.g., at least 60%, at least 70%, at least 80%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% identity to SEQ ID NO: 13 herein can be found in the “Enzymes”section below.

The compositions may be prepared in accordance with methods known in theart and may be in the form of a liquid or a dry composition. Thecompositions may be stabilized in accordance with methods known in theart.

Examples are given below of preferred uses of the compositions of thepresent invention. The dosage of the composition and other conditionsunder which the composition is used may be determined on the basis ofmethods known in the art.

Processes of the Invention

Producing a fermentation product from Gelatinized Starch Material Usinga Trehalase of the Invention

In this aspect the present invention relates to producing a fermentationproduct, in particular ethanol, from gelatinized and/or ungelatinizedstarch-containing material or cellulosic material. Fermentable sugarsgenerated during saccharification/hydrolysis are converted to thedesired fermentation in question, in particular ethanol, duringfermentation by a fermenting organism, in particular yeast.

In an embodiment the invention relates to processes of producing afermentation product, in particular ethanol, comprising

-   -   (a) liquefying a starch-containing material with an        alpha-amylase;    -   optionally pre-saccharifying the liquefied material before step        (b);    -   (b) saccharifying the liquefied material;    -   (c) fermenting using a fermentation organism;    -   wherein        -   i) a glucoamylase;        -   ii) a trehalase of the invention;        -   iii) optionally a cellulolytic enzyme composition and/or a            protease;            are present and/or added during    -   saccharification step (b);    -   fermentation step (c);    -   simultaneous saccharification and fermentation;    -   optionally presaccharification step before step (b).        Liquefaction Step (a)

According to processes of the invention, liquefaction in step (a) iscarried out by subjecting starch-containing material at a temperatureabove the initial gelatinization temperature, in particular at atemperature between 80-90° C., to an alpha-amylase and optionally aprotease and other enzymes, such as a glucoamylase, a pullulanase and/ora phytase.

The term “initial gelatinization temperature” means the lowesttemperature at which gelatinization of the starch-containing materialcommences. In general, starch heated in water begins to gelatinizebetween about 50° C. and 75° C.; the exact temperature of gelatinizationdepends on the specific starch and can readily be determined by theskilled artisan. Thus, the initial gelatinization temperature may varyaccording to the plant species, to the particular variety of the plantspecies as well as with the growth conditions. In the context of thisinvention the initial gelatinization temperature of a givenstarch-containing material may be determined as the temperature at whichbirefringence is lost in 5% of the starch granules using the methoddescribed by Gorinstein and Lii, 1992, Starch/Starke 44(12): 461-466.

According to the invention liquefaction in step (a) is typically carriedout at a temperature in the range from 70-100° C. In an embodiment thetemperature in liquefaction is between 75-95° C., such as between 75-90°C., preferably between 80-90° C., such as 82-88° C., such as around 85°C.

The pH in liquefaction may be in the range between 3 and 7, preferablyfrom 4 to 6, or more preferably from 4.5 to 5.5.

According to the invention a jet-cooking step may be carried out priorto liquefaction in step (a). The jet-cooking may be carried out at atemperature between 110-145° C., preferably 120-140° C., such as125-135° C., preferably around 130° C. for about 1-15 minutes,preferably for about 3-10 minutes, especially around about 5 minutes.

In an embodiment, the process of the invention further comprises, priorto the liquefaction step (a), the steps of:

x) reducing the particle size of the starch-containing material,preferably by dry milling;

z) forming a slurry comprising the starch-containing material and water.

According to the invention the dry solid content (DS) in liquefactionlies in the range from 20-55 wt.-%, preferably 25-45 wt.-%, morepreferably 30-40 wt.-% or 30-45 wt-%.

The starch-containing starting material, such as whole grains, may bereduced in particle size, e.g., by milling, in order to open up thestructure, to increase surface area, and allowing for furtherprocessing. Generally there are two types of processes: wet and drymilling. In dry milling whole kernels are milled and used. Wet millinggives a good separation of germ and meal (starch granules and protein).Wet milling is often applied at locations where the starch hydrolysateis used in production of, e.g., syrups. Both dry milling and wet millingare well known in the art of starch processing. According to the presentinvention dry milling is preferred.

In an embodiment the particle size is reduced to between 0.05 to 3.0 mm,preferably 0.1-0.5 mm, or so that at least 30%, preferably at least 50%,more preferably at least 70%, even more preferably at least 90% of thestarch-containing material fit through a sieve with a 0.05 to 3.0 mmscreen, preferably 0.1-0.5 mm screen. In another embodiment at least50%, preferably at least 70%, more preferably at least 80%, especiallyat least 90% of the starch-containing material fit through a sieve with#6 screen.

Liquefaction in step (a) may be carried out for 0.5-5 hours, such as 1-3hours, such as typically around 2 hours.

The alpha-amylase and other optional enzymes, such as protease, mayinitially be added to the aqueous slurry to initiate liquefaction(thinning). In an embodiment only a portion of the enzymes (e.g., about⅓) is added to the aqueous slurry, while the rest of the enzymes (e.g.,about ⅔) are added in liquefaction step (a).

A non-exhaustive list of examples of alpha-amylases can be found belowin the “Alpha-Amylase Present and/or Added In Liquefaction”-section. Ina preferred embodiment the alpha-amylase is a bacterial alpha-amylase.Bacterial alpha-amylases are typically thermostable. In a preferredembodiment the alpha-amylase is derived from the genus Bacillus, such asa strain of Bacillus stearothermophilus, in particular a variant of aBacillus stearothermophilus alpha-amylase, such as the one shown in SEQID NO: 3 in WO 99/019467 or SEQ ID NO: 18 herein.

In an embodiment the alpha-amylase used in liquefaction step (a) is avariant of the Bacillus stearothermophilus alpha-amylase shown in SEQ IDNO: 18 herein, in particular with the double deletions in I181*+G182*,and optionally with a N193F substitution, and truncated to be around 491amino acids long, e.g., from 480-495 amino acids long.

Examples of suitable Bacillus stearothermophilus alpha-amylase variantscan be found below in the “Thermostable Alpha-Amylase”-section andinclude one from the following group of Bacillus stearothermophilusalpha-amylase variants with double deletions I181*+G182*, and optionallysubstitution N193F, and additionally the following substitutions:

E129V+K177L+R179E;

V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;

V59A+Q89R+E129V+K177L+R179E+Q254S+M284V;

V59A+E129V+K177L+R179E+Q254S+M284V; and

E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQ ID NO: 18 fornumbering).

According to processes of the invention, liquefaction in step (a) may becarried out using a combination of alpha-amylase (e.g., Bacillusstearothermophilus alpha-amylase shown in SEQ ID NO: 18) and protease(e.g., Pyrococcus furiosus (pfu protease) shown in SEQ ID NO: 22). Aglucoamylase may also be present, such as the one derived fromPenicillium oxalicum shown in SEQ ID NO: 23 herein (see the“Glucoamylase Present and/or Added In Liquefaction Step (a)”-sectionbelow.

Saccharification and Fermentation

A trehalase of the invention, a glucoamylase and optionally a proteaseand/or a cellulolytic enzyme composition may be present and/or added insaccharification step (b); fermentation step (c); simultaneoussaccharification and fermentation (SSF); optionally apresaccharification step before step (b).

In a preferred embodiment the glucoamylase is added together with afungal alpha-amylase, in particular acid fungal alpha-amylase. Examplesof glucoamylases can be found in the “Glucoamylases Present and/or AddedIn Saccharification and/or Fermentation”-section below.

When doing sequential saccharification and fermentation,saccharification step (b) may be carried out at conditions well-known inthe art, i.e., suitable for enzyme saccharification. For instance, thesaccharification step (b) may last up to from about 24 to about 72hours.

In an embodiment pre-saccharification is done before saccharification instep (b). Pre-saccharification is typically done for 40-90 minutes at atemperature between 30-65° C., typically about 60° C.Pre-saccharification is in an embodiment followed by saccharificationduring fermentation in simultaneous saccharification and fermentation(SSF). Saccharification is typically carried out at temperatures from20-75° C., preferably from 40-70° C., typically around 60° C., and at apH between 4 and 5, normally at about pH 4.5.

Simultaneous saccharification and fermentation (“SSF”) is widely used inindustrial scale fermentation product production processes, especiallyethanol production processes. When doing SSF the saccharification step(b) and the fermentation step (c) are carried out simultaneously. Thereis no holding stage for the saccharification, meaning that a fermentingorganism, in particular yeast, and enzymes, may be added together.However, it is also contemplated to add the fermenting organism andenzymes separately. SSF is according to the invention typically carriedout at a temperature from 25° C. to 40° C., such as from 28° C. to 35°C., such as from 30° C. to 34° C., preferably around about 32° C. In anembodiment fermentation is ongoing for 6 to 120 hours, in particular 24to 96 hours. In an embodiment the pH is between 4-5.

In an embodiment of the invention a cellulolytic composition is presentand/or added in saccharification step (b), fermentation step (c) orsimultaneous saccharification and fermentation (SSF) orpre-saccharification before step (b). Examples of such cellulolyticcompositions can be found in the “Cellulolytic Enzyme Compositionpresent and/or added during Saccharification and/orFermentation”-section below. The optional cellulolytic enzymecomposition may be present and/or added together with the glucoamylaseand trehalase of the invention. Examples of proteases can be found inthe “Proteases Present and/or Added In Saccharification and/orFermentation”-section below.

In a preferred embodiment the trehalase is present and/or added in anamount between 0.01-20 ug EP trehalase/g DS, such as between 0.05-15 ugEP terhalase/g DS, such as between 0.5 and 10 ug EP trehalase/g DS.

Starch-Containing Materials

According to the invention any suitable starch-containing startingmaterial may be used. The starting material is generally selected basedon the desired fermentation product, in particular ethanol. Examples ofstarch-containing starting materials, suitable for use in processes ofthe present invention, include cereal, tubers or grains. Specificallythe starch-containing material may be corn, wheat, barley, rye, milo,sago, cassava, tapioca, sorghum, oat, rice, peas, beans, or sweetpotatoes, or mixtures thereof. Contemplated are also waxy and non-waxytypes of corn and barley.

In a preferred embodiment the starch-containing starting material iscorn.

In a preferred embodiment the starch-containing starting material iswheat.

In a preferred embodiment the starch-containing starting material isbarley.

In a preferred embodiment the starch-containing starting material isrye.

In a preferred embodiment the starch-containing starting material ismilo.

In a preferred embodiment the starch-containing starting material issago.

In a preferred embodiment the starch-containing starting material iscassava.

In a preferred embodiment the starch-containing starting material istapioca.

In a preferred embodiment the starch-containing starting material issorghum.

In a preferred embodiment the starch-containing starting material isrice,

In a preferred embodiment the starch-containing starting material ispeas.

In a preferred embodiment the starch-containing starting material isbeans.

In a preferred embodiment the starch-containing starting material issweet potatoes.

In a preferred embodiment the starch-containing starting material isoats.

Producing a Fermentation Product from Ungelatinized Starch MaterialUsing a Trehalase of the Invention

A trehalase of the invention may suitably be used in a raw starchhydrolysis (RSH) process for producing desired fermentation products, inparticular ethanol. In RSH processes the starch does not gelatinize asthe process is carried out at temperatures below the initialgelatinization temperature of the starch in question (defined above).

The desired fermentation product may in an embodiment be ethanolproduced from ungelatinized (i.e., uncooked), preferably milled, grains,such as corn, or small grains such as wheat, oats, barley, rye, rice, orcereals such as sorghum. Examples of suitable starch-containing startingmaterials are listed in the section “Starch-ContainingMaterials”-section above.

Accordingly, in this aspect the invention relates to processes ofproducing fermentation products from starch-containing materialcomprising:

(a) saccharifying a starch-containing material at a temperature belowthe initial gelatinization temperature; and

(b) fermenting using a fermentation organism; and

(c) optionally recovering the fermentation product;

wherein saccharification and/or fermentation is done in the presence ofthe following enzymes: glucoamylase, alpha-amylase, trehalase of theinvention, and optionally a cellulolytic enzyme composition and/or aprotease.

Before step (a) an aqueous slurry of starch-containing material, such asgranular starch, having 10-55 wt.-% dry solids (DS), preferably 25-45wt.-% dry solids, more preferably 30-40% dry solids of starch-containingmaterial may be prepared. The slurry may include water and/or processwaters, such as stillage (backset), scrubber water, evaporatorcondensate or distillate, side-stripper water from distillation, orprocess water from other fermentation product plants.

Because a raw starch hydrolysis process of the invention is carried outbelow the initial gelatinization temperature, and thus no significantviscosity increase takes place, high levels of stillage may be used, ifdesired. In an embodiment the aqueous slurry contains from about 1 toabout 70 vol.-%, preferably 15-60% vol.-%, especially from about 30 to50 vol.-% water and/or process waters, such as stillage (backset),scrubber water, evaporator condensate or distillate, side-stripper waterfrom distillation, or process water from other fermentation productplants, or combinations thereof, or the like.

In an embodiment backset, or another recycled stream, is added to theslurry before step (a), or to the saccharification (step (a)), or to thesimultaneous saccharification and fermentation steps (combined step (a)and step (b)).

A RSH process of the invention is conducted at a temperature below theinitial gelatinization temperature, which means that the temperature atwhich a separate step (a) is carried out typically lies in the rangebetween 25-75° C., such as between 30-70° C., or between 45-60° C.

In a preferred embodiment the temperature during fermentation in step(b) or simultaneous saccharification and fermentation in steps (a) and(b) is between 25° C. and 40° C., preferably between 28° C. and 36° C.,such as between 28° C. and 35° C., such as between 28° C. and 34° C.,such as around 32° C.

In an embodiment of the invention fermentation is carried out for 30 to150 hours, preferably 48 to 96 hours. 66.

In an embodiment fermentation is carried out so that the sugar level,such as glucose level, is kept at a low level, such as below 6 wt.-%,such as below about 3 wt.-%, such as below about 2 wt.-%, such as belowabout 1 wt.-%, such as below about 0.5%, or below 0.25% wt.-%, such asbelow about 0.1 wt.-%. Such low levels of sugar can be accomplished bysimply employing adjusted quantities of enzymes and fermenting organism.A skilled person in the art can easily determine which doses/quantitiesof enzyme and fermenting organism to use. The employed quantities ofenzyme and fermenting organism may also be selected to maintain lowconcentrations of maltose in the fermentation broth. For instance, themaltose level may be kept below about 0.5 wt.-%, such as below about 0.2wt.-%.

The process of the invention may be carried out at a pH from 3 and 7,preferably from 3 to 6, or more preferably from 3.5 to 5.0.

The term “granular starch” means raw uncooked starch, i.e., starch inits natural form found in, e.g., cereal, tubers or grains. Starch isformed within plant cells as tiny granules insoluble in water. When putin cold water, the starch granules may absorb a small amount of theliquid and swell. At temperatures up to around 50° C. to 75° C. theswelling may be reversible. However, at higher temperatures anirreversible swelling called “gelatinization” begins. The granularstarch may be a highly refined starch, preferably at least 90%, at least95%, at least 97% or at least 99.5% pure, or it may be a more crudestarch-containing materials comprising (e.g., milled) whole grainsincluding non-starch fractions such as germ residues and fibers.

The raw material, such as whole grains, may be reduced in particle size,e.g., by milling, in order to open up the structure and allowing forfurther processing. Examples of suitable particle sizes are disclosed inU.S. Pat. No. 4,514,496 and WO2004/081193 (both references areincorporated by reference). Two processes are preferred according to theinvention: wet and dry milling. In dry milling whole kernels are milledand used. Wet milling gives a good separation of germ and meal (starchgranules and protein) and is often applied at locations where the starchhydrolysate is used in production of, e.g., syrups. Both dry and wetmilling is well known in the art of starch processing.

In an embodiment the particle size is reduced to between 0.05 to 3.0 mm,preferably 0.1-0.5 mm, or so that at least 30%, preferably at least 50%,more preferably at least 70%, even more preferably at least 90% of thestarch-containing material fit through a sieve with a 0.05 to 3.0 mmscreen, preferably 0.1-0.5 mm screen. In a preferred embodimentstarch-containing material is prepared by reducing the particle size ofthe starch-containing material, preferably by milling, such that atleast 50% of the starch-containing material has a particle size of0.1-0.5 mm.

In a preferred embodiment the trehalase is present and/or added in anamount between 0.01-20 ug EP trehalase/g DS, such as between 0.05-15 ugEP terhalase/g DS, such as between 0.5 and 10 ug EP trehalase/g DS.

According to the invention the enzymes are added so that theglucoamylase is present in an amount of 0.001 to 10 AGU/g DS, preferablyfrom 0.01 to 5 AGU/g DS, especially 0.1 to 0.5 AGU/g DS.

According to the invention the enzymes are added so that thealpha-amylase is present or added in an amount of 0.001 to 10 AFAU/g DS,preferably from 0.01 to 5 AFAU/g DS, especially 0.3 to 2 AFAU/g DS or0.001 to 1 FAU-F/g DS, preferably 0.01 to 1 FAU-F/g DS.

According to the invention the enzymes are added so that thecellulolytic enzyme composition is present or added in an amount1-10,000 micro grams EP/g DS, such as 2-5,000, such as 3 and 1,000, suchas 4 and 500 micro grams EP/g DS.

According to the invention the enzymes are added so that thecellulolytic enzyme composition is present or added in an amount in therange from 0.1-100 FPU per gram total solids (TS), preferably 0.5-50 FPUper gram TS, especially 1-20 FPU per gram TS.

In an embodiment of the invention the enzymes are added so that theprotease is present in an amount of 0.0001-1 mg enzyme protein per g DS,preferably 0.001 to 0.1 mg enzyme protein per g DS. Alternatively, theprotease is present and/or added in an amount of 0.0001 to 1 LAPU/g DS,preferably 0.001 to 0.1 LAPU/g DS and/or 0.0001 to 1 mAU-RH/g DS,preferably 0.001 to 0.1 mAU-RH/g DS.

In an embodiment of the invention the enzymes are added so that theprotease is present or added in an amount in the range 1-1,000 μg EP/gDS, such as 2-500 μg EP/g DS, such as 3-250 μg EP/g DS.

In a preferred embodiment ratio between glucoamylase and alpha-amylaseis between 99:1 and 1:2, such as between 98:2 and 1:1, such as between97:3 and 2:1, such as between 96:4 and 3:1, such as 97:3, 96:4, 95:5,94:6, 93:7, 90:10, 85:15, 83:17 or 65:35 (mg EP glucoamylase: mg EPalpha-amylase).

In a preferred embodiment the total dose of glucoamylase andalpha-amylase is according to the invention from 10-1,000 μg/g DS, suchas from 50-500 μg/g DS, such as 75-250 μg/g DS.

In a preferred embodiment the total dose of cellulolytic enzymecomposition added is from 10-500 μg/g DS, such as from 20-400 μg/g DS,such as 20-300 μg/g DS.

In an embodiment the glucoamylase, such as one derived from Trametescingulata, used in fermentation or SSF exhibits at least 60%, such as atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% oreven 100% identity to the mature part of SEQ ID NO: 14.

In an embodiment the glucoamylase, such as one derived from Pycnoporussanguineus, used in fermentation or SSF exhibits at least 60%, such asat least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% oreven 100% identity to the mature part of SEQ ID NO: 28.

In an embodiment the alpha-amylase used in fermentation or SSF exhibitsat least 60%, such as at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or even 100% identity to the mature part of SEQ ID NO:15.

In a preferred embodiment the invention relates to processes ofproducing fermentation products from starch-containing materialcomprising:

(a) saccharifying a starch-containing material at a temperature belowthe initial gelatinization temperature; and

(b) fermenting using a fermentation organism;

wherein saccharification and/or fermentation is done in the presence ofthe following enzymes:

-   -   i) glucoamylase;    -   ii) alpha-amylase;    -   iii) trehalse of the invention;    -   iii) optionally a cellulolytic enzyme composition and/or a        protease.

In a preferred embodiment the enzymes may be added as an enzymecomposition of the invention. In a preferred embodiment steps (a) and(b) are carried out simultaneously (i.e., one-step fermentation).However, step (a) and (b) may also be carried our sequentially.

Fermentation

Fermentation is carried out in a fermentation medium. The fermentationmedium includes the fermentation substrate, that is, the carbohydratesource that is metabolized by the fermenting organism. According to theinvention the fermentation medium may comprise nutrients and growthstimulator(s) for the fermenting organism(s). Nutrient and growthstimulators are widely used in the art of fermentation and includenitrogen sources, such as ammonia; urea, vitamins and minerals, orcombinations thereof.

Fermenting Organisms for Starch Based Fermentation

The term “fermenting organism” refers to any organism, includingbacterial and fungal organisms, especially yeast, suitable for use in afermentation process and capable of producing the desired fermentationproduct. Especially suitable fermenting organisms are able to ferment,i.e., convert, sugars, such as glucose or maltose, directly orindirectly into the desired fermentation product, such as in particularethanol. Examples of fermenting organisms include fungal organisms, suchas in particular yeast. Preferred yeast includes strains ofSaccharomyces spp., in particular, Saccharomyces cerevisiae.

Suitable concentrations of the viable fermenting organism duringfermentation, such as SSF, are well known in the art or can easily bedetermined by the skilled person in the art. In one embodiment thefermenting organism, such as ethanol fermenting yeast, (e.g.,Saccharomyces cerevisiae) is added to the fermentation medium so thatthe viable fermenting organism, such as yeast, count per mL offermentation medium is in the range from 10⁵ to 10¹², preferably from10⁷ to 10¹⁰, especially about 5×10⁷.

Examples of commercially available yeast includes, e.g., RED START™ andETHANOL RED™ yeast (available from Fermentis/Lesaffre, USA), FALI(available from Fleischmann's Yeast, USA), SUPERSTART and THERMOSACC™fresh yeast (available from Ethanol Technology, WI, USA), BIOFERM AFTand XR (available from NABC—North American Bioproducts Corporation, GA,USA), GERT STRAND (available from Gert Strand AB, Sweden), and FERMIOL(available from DSM Specialties).

Recovery

Subsequent to fermentation, e.g., SSF, the fermentation product, inparticular ethanol may be separated from the fermentation medium. Theslurry may be distilled to recover/extract the desired fermentationproduct (i.e., ethanol). Alternatively the desired fermentation product(i.e., ethanol) may be extracted from the fermentation medium by microor membrane filtration techniques. The fermentation product (i.e.,ethanol) may also be recovered by stripping or other method well knownin the art.

Alpha-Amylase Present and/or Added in Liquefaction

According to the invention an alpha-amylase is present and/or added inliquefaction optionally together with other enzymes such as a protease,a glucoamylase, phytase and/or pullulanase.

The alpha-amylase added in liquefaction step (a) may be anyalpha-amylase. Preferred are bacterial alpha-amylases, which typicallyare stable at temperatures used in liquefaction.

Bacterial Alpha-Amylase

The term “bacterial alpha-amylase” means any bacterial alpha-amylaseclassified under EC 3.2.1.1. A bacterial alpha-amylase used according tothe invention may, e.g., be derived from a strain of the genus Bacillus,which is sometimes also referred to as the genus Geobacillus. In anembodiment the Bacillus alpha-amylase is derived from a strain ofBacillus amyloliquefaciens, Bacillus licheniformis, Bacillusstearothermophilus, or Bacillus subtilis, but may also be derived fromother Bacillus sp.

Specific examples of bacterial alpha-amylases include the Bacillusstearothermophilus alpha-amylase of SEQ ID NO: 3 in WO 99/19467 or SEQID NO: 18 herein, the Bacillus amyloliquefaciens alpha-amylase of SEQ IDNO: 5 in WO 99/19467, and the Bacillus licheniformis alpha-amylase ofSEQ ID NO: 4 in WO 99/19467 (all sequences are hereby incorporated byreference). In an embodiment the alpha-amylase may be an enzyme having adegree of identity of at least 60%, e.g., at least 70%, at least 80%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% to any of the sequences shown in SEQ ID NOS: 3, 4 or 5,respectively, in WO 99/19467.

In an embodiment the alpha-amylase may be an enzyme having a degree ofidentity of at least 60%, e.g., at least 70%, at least 80%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% to any ofthe sequences shown in SEQ ID NO: 3 in WO 99/19467 or SEQ ID NO: 18herein.

In a preferred embodiment the alpha-amylase is derived from Bacillusstearothermophilus. The Bacillus stearothermophilus alpha-amylase may bea mature wild-type or a mature variant thereof. The mature Bacillusstearothermophilus alpha-amylases may naturally be truncated duringrecombinant production. For instance, the Bacillus stearothermophilusalpha-amylase may be a truncated so it has around 491 amino acids, e.g.,so that it is between 480-495 amino acids long, so it lacks a functionalstarch binding domain (compared to SEQ ID NO: 3 in WO 99/19467) or SEQID NO: 18 herein.

The Bacillus alpha-amylase may also be a variant and/or hybrid. Examplesof such a variant can be found in any of WO 96/23873, WO 96/23874, WO97/41213, WO 99/19467, WO 00/60059, and WO 02/10355 (all documents arehereby incorporated by reference). Specific alpha-amylase variants aredisclosed in U.S. Pat. Nos. 6,093,562, 6,187,576, 6,297,038, and7,713,723 (hereby incorporated by reference) and include Bacillusstearothermophilus alpha-amylase (often referred to as BSGalpha-amylase) variants having a deletion of one or two amino acids atpositions R179, G180, 1181 and/or G182, preferably a double deletiondisclosed in WO 96/23873—see, e.g., page 20, lines 1-10 (herebyincorporated by reference), preferably corresponding to deletion ofpositions 1181 and G182 compared to the amino acid sequence of Bacillusstearothermophilus alpha-amylase set forth in SEQ ID NO: 3 disclosed inWO 99/19467 or SEQ ID NO: 1 herein or the deletion of amino acids R179and G180 using SEQ ID NO: 3 in WO 99/19467 or SEQ ID NO: 1 herein fornumbering (which reference is hereby incorporated by reference). Evenmore preferred are Bacillus alpha-amylases, especially Bacillusstearothermophilus alpha-amylases, which have a double deletioncorresponding to a deletion of positions 181 and 182 and furthercomprise a N193F substitution (also denoted I181*+G182*+N193F) comparedto the wild-type BSG alpha-amylase amino acid sequence set forth in SEQID NO: 3 disclosed in WO 99/19467 or SEQ ID NO: 18 herein. The bacterialalpha-amylase may also have a substitution in a position correspondingto S239 in the Bacillus licheniformis alpha-amylase shown in SEQ ID NO:4 in WO 99/19467, or a S242 and/or E188P variant of the Bacillusstearothermophilus alpha-amylase of SEQ ID NO: 3 in WO 99/19467 or SEQID NO: 18 herein.

In an embodiment the variant is a S242A, E or Q variant, preferably aS242Q variant, of the Bacillus stearothermophilus alpha-amylase (usingSEQ ID NO: 18 herein for numbering).

In an embodiment the variant is a position E188 variant, preferablyE188P variant of the Bacillus stearothermophilus alpha-amylase (usingSEQ ID NO: 18 herein for numbering).

The bacterial alpha-amylase may in an embodiment be a truncated Bacillusalpha-amylase. Especially the truncation is so that, e.g., the Bacillusstearothermophilus alpha-amylase shown in SEQ ID NO: 3 in WO 99/19467 orSEQ ID NO: 18 herein, is around 491 amino acids long, such as from 480to 495 amino acids long, or so it lack a functional starch bindingdomain.

Bacterial Hybrid Alpha-Amylases

The bacterial alpha-amylase may also be a hybrid bacterialalpha-amylase, e.g., an alpha-amylase comprising 445 C-terminal aminoacid residues of the Bacillus licheniformis alpha-amylase (shown in SEQID NO: 4 of WO 99/19467) and the 37 N-terminal amino acid residues ofthe alpha-amylase derived from Bacillus amyloliquefaciens (shown in SEQID NO: 5 of WO 99/19467). In a preferred embodiment this hybrid has oneor more, especially all, of the following substitutions:

G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S (using the Bacilluslicheniformis numbering in SEQ ID NO: 4 of WO 99/19467). Also preferredare variants having one or more of the following mutations (orcorresponding mutations in other Bacillus alpha-amylases): H154Y, A181T,N190F, A209V and Q264S and/or the deletion of two residues betweenpositions 176 and 179, preferably the deletion of E178 and G179 (usingSEQ ID NO: 5 of WO 99/19467 for position numbering).

In an embodiment the bacterial alpha-amylase is the mature part of thechimeric alpha-amylase disclosed in Richardson et al. (2002), TheJournal of Biological Chemistry, Vol. 277, No 29, Issue 19 July, pp.267501-26507, referred to as BD5088 or a variant thereof. Thisalpha-amylase is the same as the one shown in SEQ ID NO: 2 in WO2007134207. The mature enzyme sequence starts after the initial “Met”amino acid in position 1.

Thermostable Alpha-Amylase

The alpha-amylase may be a thermostable alpha-amylase, such as athermostable bacterial alpha-amylase, preferably from Bacillusstearothermophilus.

In an embodiment of the invention the alpha-amylase is an bacterialalpha-amylase, preferably derived from the genus Bacillus, especially astrain of Bacillus stearothermophilus, in particular the Bacillusstearothermophilus as disclosed in WO 99/019467 as SEQ ID NO: 3 (SEQ IDNO: 18 herein) with one or two amino acids deleted at positions R179,G180, I181 and/or G182, in particular with R179 and G180 deleted, orwith 1181 and G182 deleted, with mutations in below list of mutations.

In preferred embodiments the Bacillus stearothermophilus alpha-amylaseshave double deletion 1181+G182, and optionally substitution N193F,further comprising mutations selected from below list:

-   -   V59A+Q89R+G112D+E129V+K177L+R179E+K220P+N224L+Q254S;    -   V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;    -   V59A+Q89R+E129V+K177L+R179E+K220P+N224L+Q254S+D269E+D281N;    -   V59A+Q89R+E129V+K177L+R179E+K220P+N224L+Q254S+I270L;    -   V59A+Q89R+E129V+K177L+R179E+K220P+N224L+Q254S+H274K;    -   V59A+Q89R+E129V+K177L+R179E+K220P+N224L+Q254S+Y276F;    -   V59A+E129V+R157Y+K177L+R179E+K220P+N224L+S242Q+Q254S;    -   V59A+E129V+K177L+R179E+H208Y+K220P+N224L+S242Q+Q254S;    -   59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S;    -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+H274K;    -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+Y276F;    -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+D281N;    -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+M284T;    -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+G416V;    -   V59A+E129V+K177L+R179E+K220P+N224L+Q254S;    -   V59A+E129V+K177L+R179E+K220P+N224L+Q254S+M284T;    -   A91 L+M961+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S;    -   E129V+K177L+R179E;    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S;    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+Y276F+L427M;    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+M284T;    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+N376*+I377*;    -   E129V+K177L+R179E+K220P+N224L+Q254S;    -   E129V+K177L+R179E+K220P+N224L+Q254S+M284T;    -   E129V+K177L+R179E+S242Q;    -   E129V+K177L+R179V+K220P+N224L+S242Q+Q254S;    -   K220P+N224L+S242Q+Q254S;    -   M284V;    -   V59A+Q89R+E129V+K177L+R179E+Q254S+M284V.    -   V59A+E129V+K177L+R179E+Q254S+M284V;

Specific information about the thermostability of above alpha-amylasesvariants can be found in WO12/088303 (Novozymes) which is herebyincorporated by reference.

In a preferred embodiment the alpha-amylase is selected from the groupof Bacillus stearothermophilus alpha-amylase variants having a doubledeletion in I181+G182, and optionally a substitution in N193F, andsubstitutions from the following list

-   -   E129V+K177L+R179E;    -   V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;    -   V59A+Q89R+E129V+K177L+R179E+Q254S+M284V;    -   V59A+E129V+K177L+R179E+Q254S+M284V; and    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQ ID NO: 18        herein for numbering).

It should be understood that when referring to Bacillusstearothermophilus alpha-amylase and variants thereof they are normallyproduced in truncated form. In particular, the truncation may be so thatthe Bacillus stearothermophilus alpha-amylase shown in SEQ ID NO: 3 inWO 99/19467 or SEQ ID NO: 18 herein, or variants thereof, are truncatedin the C-terminal and are typically around 491 amino acids long, such asfrom 480-495 amino acids long, or so that it lacks a functional starchbinding domain.

In a preferred embodiment the alpha-amylase variant may be an enzymehaving a degree of identity of at least 60%, e.g., at least 70%, atleast 80%, at least 90%, at least 95%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98% or at least 99%, but less than 100% to the sequence shown inSEQ ID NO: 3 in WO 99/19467 or SEQ ID NO: 18 herein.

In an embodiment the bacterial alpha-amylase, e.g., Bacillusalpha-amylase, such as especially Bacillus stearothermophilusalpha-amylase, or variant thereof, is dosed to liquefaction in aconcentration between 0.01-10 KNU-A/g DS, e.g., between 0.02 and 5KNU-A/g DS, such as 0.03 and 3 KNU-A, preferably 0.04 and 2 KNU-A/g DS,such as especially 0.01 and 2 KNU-A/g DS. In an embodiment the bacterialalpha-amylase, e.g., Bacillus alpha-amylase, such as especially Bacillusstearothermophilus alpha-amylases, or variant thereof, is dosed toliquefaction in a concentration of between 0.0001-1 mg EP (EnzymeProtein)/g DS, e.g., 0.0005-0.5 mg EP/g DS, such as 0.001-0.1 mg EP/gDS.

Protease Present and/or Added in Liquefaction

According to the invention a protease may optionally be present and/oradded in liquefaction together with the alpha-amylase, and an optionalglucoamylase, phytase and/or pullulanase.

Proteases are classified on the basis of their catalytic mechanism intothe following groups: Serine proteases (S), Cysteine proteases (C),Aspartic proteases (A), Metallo proteases (M), and Unknown, or as yetunclassified, proteases (U), see Handbook of Proteolytic Enzymes, A. J.Barrett, N. D. Rawlings, J. F. Woessner (eds), Academic Press (1998), inparticular the general introduction part.

In a preferred embodiment the thermostable protease used according tothe invention is a “metallo protease” defined as a protease belonging toEC 3.4.24 (metalloendopeptidases); preferably EC 3.4.24.39 (acid metalloproteinases).

To determine whether a given protease is a metallo protease or not,reference is made to the above “Handbook of Proteolytic Enzymes” and theprinciples indicated therein. Such determination can be carried out forall types of proteases, be it naturally occurring or wild-typeproteases; or genetically engineered or synthetic proteases.

Protease activity can be measured using any suitable assay, in which asubstrate is employed, that includes peptide bonds relevant for thespecificity of the protease in question. Assay-pH and assay-temperatureare likewise to be adapted to the protease in question. Examples ofassay-pH-values are pH 6, 7, 8, 9, 10, or 11. Examples ofassay-temperatures are 30, 35, 37, 40, 45, 50, 55, 60, 65, 70 or 80° C.

Examples of protease substrates are casein, such as Azurine-CrosslinkedCasein (AZCL-casein). Two protease assays are described below in the“Materials & Methods”-section, of which the so-called “AZCL-CaseinAssay” is the preferred assay.

There are no limitations on the origin of the protease used in a processof the invention as long as it fulfills the thermostability propertiesdefined below.

In one embodiment the protease is of fungal origin.

The protease may be a variant of, e.g., a wild-type protease as long asthe protease is thermostable. In a preferred embodiment the thermostableprotease is a variant of a metallo protease as defined above. In anembodiment the thermostable protease used in a process of the inventionis of fungal origin, such as a fungal metallo protease, such as a fungalmetallo protease derived from a strain of the genus Thermoascus,preferably a strain of Thermoascus aurantiacus, especially Thermoascusaurantiacus CGMCC No. 0670 (classified as EC 3.4.24.39).

In an embodiment the thermostable protease is a variant of the maturepart of the metallo protease shown in SEQ ID NO: 2 disclosed in WO2003/048353 or the mature part of SEQ ID NO: 1 in WO 2010/008841 andshown as SEQ ID NO: 17 herein, further with mutations selected frombelow list:

-   -   S5*+D79L+S87P+A112P+D142L;    -   D79L+S87P+A112P+T124V+D142L;    -   S5*+N26R+D79L+S87P+A112P+D142L;    -   N26R+T46R+D79L+S87P+A112P+D142L;    -   T46R+D79L+S87P+T116V+D142L;    -   D79L+P81R+S87P+A112P+D142L;    -   A27K+D79L+S87P+A112P+T124V+D142L;    -   D79L+Y82F+S87P+A112P+T124V+D142L;    -   D79L+S87P+A112P+T124V+A126V+D142L;    -   D79L+S87P+A112P+D142L;    -   D79L+Y82F+S87P+A112P+D142L;    -   S38T+D79L+S87P+A112P+A126V+D142L;    -   D79L+Y82F+S87P+A112P+A126V+D142L;    -   A27K+D79L+S87P+A112P+A126V+D142L;    -   D79L+S87P+N98C+A112P+G135C+D142L;    -   D79L+S87P+A112P+D142L+T141C+M161C;    -   S36P+D79L+S87P+A112P+D142L;    -   A37P+D79L+S87P+A112P+D142L;    -   S49P+D79L+S87P+A112P+D142L;    -   S50P+D79L+S87P+A112P+D142L;    -   D79L+S87P+D104P+A112P+D142L;    -   D79L+Y82F+S87G+A112P+D142L;    -   S70V+D79L+Y82F+S87G+Y97W+A112P+D142L;    -   D79L+Y82F+S87G+Y97W+D104P+A112P+D142L;    -   S70V+D79L+Y82F+S87G+A112P+D142L;    -   D79L+Y82F+S87G+D104P+A112P+D142L;    -   D79L+Y82F+S87G+A112P+A126V+D142L;    -   Y82F+S87G+S70V+D79L+D104P+A112P+D142L;    -   Y82F+S87G+D79L+D104P+A112P+A126V+D142L;    -   A27K+D79L+Y82F+S87G+D104P+A112P+A126V+D142L;    -   A27K+Y82F+S87G+D104P+A112P+A126V+D142L;    -   A27K+D79L+Y82F+D104P+A112P+A126V+D142L;    -   A27K+Y82F+D104P+A112P+A126V+D142L;    -   A27K+D79L+S87P+A112P+D142L;    -   D79L+S87P+D142L.

Specific information about the thermostability of above proteasevariants can be found in WO12/088303 (Novozymes), which is herebyincorporated by reference.

In an preferred embodiment the thermostable protease is a variant of themetallo protease disclosed as the mature part of SEQ ID NO: 2 disclosedin WO 2003/048353 or the mature part of SEQ ID NO: 1 in WO 2010/008841or SEQ ID NO: 17 herein with the following mutations:

-   -   D79L+S87P+A112P+D142L;    -   D79L+S87P+D142L; or    -   A27K+D79L+Y82F+S87G+D104P+A112P+A126V+D142L.

In an embodiment the protease variant has at least 75% identitypreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, more preferably at least 91%, more preferably at least92%, even more preferably at least 93%, most preferably at least 94%,and even most preferably at least 95%, such as even at least 96%, atleast 97%, at least 98%, at least 99%, but less than 100% identity tothe mature part of the polypeptide of SEQ ID NO: 2 disclosed in WO2003/048353 or the mature part of SEQ ID NO: 1 in WO 2010/008841 or SEQID NO: 17 herein.

The thermostable protease may also be derived from any bacterium as longas the protease has the thermostability properties defined according tothe invention.

In an embodiment the thermostable protease is derived from a strain ofthe bacterium Pyrococcus, such as a strain of Pyrococcus furiosus (pfuprotease).

In an embodiment the protease is one shown as SEQ ID NO: 1 in U.S. Pat.No. 6,358,726-B1 (Takara Shuzo Company), or SEQ ID NO: 22 herein.

In another embodiment the thermostable protease is one disclosed in SEQID NO: 22 herein or a protease having at least 80% identity, such as atleast 85%, such as at least 90%, such as at least 95%, such as at least96%, such as at least 97%, such as at least 98%, such as at least 99%identity to SEQ ID NO: 1 in U.S. Pat. No. 6,358,726-B1 or SEQ ID NO: 22herein. Pyroccus furiosus protease can be purchased from Takara Bio,Japan.

Glucoamylase Present and/or Added in Liquefaction

According to the invention a glucoamylase may optionally be presentand/or added in liquefaction step (a). In a preferred embodiment theglucoamylase is added together with or separately from the alpha-amylaseand optional protease, phytase and/or pullulanase.

In a specific and preferred embodiment the glucoamylase, preferably offungal origin, preferably a filamentous fungi, is from a strain of thegenus Penicillium, especially a strain of Penicillium oxalicum, inparticular the Penicillium oxalicum glucoamylase disclosed as SEQ ID NO:2 in WO 2011/127802 (which is hereby incorporated by reference) andshown in SEQ ID NO: 23 herein.

In an embodiment the glucoamylase has at least 80%, more preferably atleast 85%, more preferably at least 90%, more preferably at least 91%,more preferably at least 92%, even more preferably at least 93%, mostpreferably at least 94%, and even most preferably at least 95%, such aseven at least 96%, at least 97%, at least 98%, at least 99% or 100%identity to the mature polypeptide shown in SEQ ID NO: 2 in WO2011/127802 or SEQ ID NO: 23 herein.

In a preferred embodiment the glucoamylase is a variant of thePenicillium oxalicum glucoamylase disclosed as SEQ ID NO: 2 in WO2011/127802 and shown in SEQ ID NO: 23 herein, having a K79Vsubstitution (using the mature sequence shown in SEQ ID NO: 23 hereinfor numbering). The K79V glucoamylase variant has reduced sensitivity toprotease degradation relative to the parent as disclosed in WO2013/036526 (which is hereby incorporated by reference).

In an embodiment the glucoamylase is derived from Penicillium oxalicum.

In an embodiment the glucoamylase is a variant of the Penicilliumoxalicum glucoamylase disclosed as SEQ ID NO: 2 in WO 2011/127802 andshown in SEQ ID NO: 23 herein. In a preferred embodiment the Penicilliumoxalicum glucoamylase is the one disclosed as SEQ ID NO: 2 in WO2011/127802 and shown in SEQ ID NO: 23 herein having Val (V) in position79 (using SEQ ID NO: 23 herein for numbering).

Contemplated Penicillium oxalicum glucoamylase variants are disclosed inWO 2013/053801 which is hereby incorporated by reference.

In an embodiment these variants have reduced sensitivity to proteasedegradation. degradation.

In an embodiment these variant have improved thermostability compared tothe parent.

More specifically, in an embodiment the glucoamylase has a K79Vsubstitution (using SEQ ID NO: 23 herein for numbering), (PE001variant), and further comprises at least one of the followingsubstitutions or combination of substitutions:

-   -   T65A; or    -   Q327F; or    -   E501V; or    -   Y504T; or    -   Y504*; or    -   T65A+Q327F; or    -   T65A+E501V; or    -   T65A+Y504T; or    -   T65A+Y504*; or    -   Q327F+E501V; or    -   Q327F+Y504T; or    -   Q327F+Y504*; or    -   E501V+Y504T; or    -   E501V+Y504*; or    -   T65A+Q327F+E501V; or    -   T65A+Q327F+Y504T; or    -   T65A+E501V+Y504T; or    -   Q327F+E501V+Y504T; or    -   T65A+Q327F+Y504*; or    -   T65A+E501V+Y504*; or    -   Q327F+E501V+Y504*; or    -   T65A+Q327F+E501V+Y504T; or    -   T65A+Q327F+E501V+Y504*;    -   E501V+Y504T; or    -   T65A+K161S; or    -   T65A+Q405T; or    -   T65A+Q327W; or    -   T65A+Q327F; or    -   T65A+Q327Y; or    -   P11F+T65A+Q327F; or    -   R1K+D3W+K5Q+G7V+N8S+T10K+P11S+T65A+Q327F; or    -   P2N+P4S+P11F+T65A+Q327F; or    -   P11F+D26C+K330+T65A+Q327F; or    -   P2N+P4S+P11F+T65A+Q327W+E501V+Y504T; or    -   R1E+D3N+P4G+G6R+G7A+N8A+T10D+P11D+T65A+Q327F; or    -   P11F+T65A+Q327W; or    -   P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or    -   P11F+T65A+Q327W+E501V+Y504T; or    -   T65A+Q327F+E501V+Y504T; or    -   T65A+S105P+Q327W; or    -   T65A+S105P+Q327F; or    -   T65A+Q327W+S364P; or    -   T65A+Q327F+S364P; or    -   T65A+S103N+Q327F; or    -   P2N+P4S+P11F+K34Y+T65A+Q327F; or    -   P2N+P4S+P11F+T65A+Q327F+D445N+V447S; or    -   P2N+P4S+P11F+T65A+I172V+Q327F; or    -   P2N+P4S+P11F+T65A+Q327F+N502*; or    -   P2N+P4S+P11F+T65A+Q327F+N502T+P563S+K571E; or    -   P2N+P4S+P11F+R31S+K33V+T65A+Q327F+N564D+K571S; or    -   P2N+P4S+P11F+T65A+Q327F+S377T; or    -   P2N+P4S+P11F+T65A+V325T+Q327W; or    -   P2N+P4S+P11F+T65A+Q327F+D445N+V447S+E501V+Y504T; or    -   P2N+P4S+P11F+T65A+I172V+Q327F+E501V+Y504T; or    -   P2N+P4S+P11F+T65A+Q327F+S377T+E501V+Y504T; or    -   P2N+P4S+P11F+D26N+K34Y+T65A+Q327F; or    -   P2N+P4S+P11F+T65A+Q327F+I375A+E501V+Y504T; or    -   P2N+P4S+P11F+T65A+K218A+K221D+Q327F+E501V+Y504T; or    -   P2N+P4S+P11F+T65A+S103N+Q327F+E501V+Y504T; or    -   P2N+P4S+T10D+T65A+Q327F+E501V+Y504T; or    -   P2N+P4S+F12Y+T65A+Q327F+E501V+Y504T; or    -   K5A+P11F+T65A+Q327F+E501V+Y504T; or    -   P2N+P4S+T10E+E18N+T65A+Q327F+E501V+Y504T; or    -   P2N+T10E+E18N+T65A+Q327F+E501V+Y504T; or    -   P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T568N; or    -   P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+K524T+G526A; or    -   P2N+P4S+P11F+K34Y+T65A+Q327F+D445N+V447S+E501V+Y504T; or    -   P2N+P4S+P11F+R31S+K33V+T65A+Q327F+D445N+V447S+E501V+Y504T; or    -   P2N+P4S+P11F+D26N+K34Y+T65A+Q327F+E501V+Y504T; or    -   P2N+P4S+P11F+T65A+F80*+Q327F+E501V+Y504T; or    -   P2N+P4S+P11F+T65A+K112S+Q327F+E501V+Y504T; or    -   P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T516P+K524T+G526A; or    -   P2N+P4S+P11F+T65A+Q327F+E501V+N502T+Y504*; or    -   P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or    -   P2N+P4S+P11F+T65A+S103N+Q327F+E501V+Y504T; or    -   K5A+P11F+T65A+Q327F+E501V+Y504T; or    -   P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T516P+K524T+G526A; or    -   P2N+P4S+P11F+T65A+V79A+Q327F+E501V+Y504T; or    -   P2N+P4S+P11F+T65A+V79G+Q327F+E501V+Y504T; or    -   P2N+P4S+P11F+T65A+V791+Q327F+E501V+Y504T; or    -   P2N+P4S+P11F+T65A+V79L+Q327F+E501V+Y504T; or    -   P2N+P4S+P11F+T65A+V79S+Q327F+E501V+Y504T; or    -   P2N+P4S+P11F+T65A+L72V+Q327F+E501V+Y504T; or    -   S255N+Q327F+E501V+Y504T; or    -   P2N+P4S+P11F+T65A+E74N+V79K+Q327F+E501V+Y504T; or    -   P2N+P4S+P11F+T65A+G220N+Q327F+E501V+Y504T; or    -   P2N+P4S+P11F+T65A+Y245N+Q327F+E501V+Y504T; or    -   P2N+P4S+P11F+T65A+Q253N+Q327F+E501V+Y504T; or    -   P2N+P4S+P11F+T65A+D279N+Q327F+E501V+Y504T; or    -   P2N+P4S+P11F+T65A+Q327F+S359N+E501V+Y504T; or    -   P2N+P4S+P11F+T65A+Q327F+D370N+E501V+Y504T; or    -   P2N+P4S+P11F+T65A+Q327F+V460S+E501V+Y504T; or    -   P2N+P4S+P11F+T65A+Q327F+V460T+P468T+E501V+Y504T; or    -   P2N+P4S+P11F+T65A+Q327F+T463N+E501V+Y504T; or    -   P2N+P4S+P11F+T65A+Q327F+S465N+E501V+Y504T; or    -   P2N+P4S+P11F+T65A+Q327F+T477N+E501V+Y504T.

In a preferred embodiment the Penicillium oxalicum glucoamylase varianthas a K79V substitution (using SEQ ID NO: 23 herein for numbering),corresponding to the PE001 variant, and further comprises one of thefollowing mutations:

-   -   P11F+T65A+Q327F; or    -   P2N+P4S+P11F+T65A+Q327F; or    -   P11F+D26C+K33C+T65A+Q327F; or    -   P2N+P4S+P11F+T65A+Q327W+E501V+Y504T; or    -   P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or    -   P11F+T65A+Q327W+E501V+Y504T.

The glucoamylase may be added in amounts from 0.1-100 micrograms EP/g,such as 0.5-50 micrograms EP/g, such as 1-25 micrograms EP/g, such as2-12 micrograms EP/g DS.

Trehalase Present and/or Added in Saccharification and/or Fermentation

According to the process of the invention a trehalase of the inventionis present and/or added during the

-   -   saccharification step (b);    -   fermentation step (c);    -   simultaneous saccharification and fermentation;    -   optionally presaccharification step before step (b).

In a preferred embodiment the mature trehalase disclosed in SEQ ID NO:30 or SEQ ID NO: 2. In a preferred embodiment the mature trehalasedisclosed in SEQ ID NO: 4. In a preferred embodiment the trehalase ispresent and/or added in an amount between 0.01-20 ug EP (Enzyme Protein)trehalase/g DS, such as between 0.05-15 ug EP terhalase/g DS, such asbetween 0.5 and 10 ug EP trehalase/g DS.

Glucoamylase Present and/or Added in Saccharification and/orFermentation

The glucoamylase present and/or added during saccharification step (b);fermentation step (c); simultaneous saccharification and fermentation;or presaccharification before step (b), may be derived from any suitablesource, e.g., derived from a microorganism or a plant. Preferredglucoamylases are of fungal or bacterial origin, selected from the groupconsisting of Aspergillus glucoamylases, in particular Aspergillus nigerG1 or G2 glucoamylase (Boel et al. (1984), EMBO J. 3 (5), p. 1097-1102),or variants thereof, such as those disclosed in WO 92/00381, WO 00/04136and WO 01/04273 (from Novozymes, Denmark); the A. awamori glucoamylasedisclosed in WO 84/02921, Aspergillus oryzae glucoamylase (Agric. Biol.Chem. (1991), 55 (4), p. 941-949), or variants or fragments thereof.Other Aspergillus glucoamylase variants include variants with enhancedthermal stability: G137A and G139A (Chen et al. (1996), Prot. Eng. 9,499-505); D257E and D293E/Q (Chen et al. (1995), Prot. Eng. 8, 575-582);N182 (Chen et al. (1994), Biochem. J. 301, 275-281); disulphide bonds,A246C (Fierobe et al. (1996), Biochemistry, 35, 8698-8704; andintroduction of Pro residues in position A435 and S436 (Li et al.(1997), Protein Eng. 10, 1199-1204.

Other glucoamylases include Athelia rolfsii (previously denotedCorticium rolfsii) glucoamylase (see U.S. Pat. No. 4,727,026 and(Nagasaka et al. (1998) “Purification and properties of theraw-starch-degrading glucoamylases from Corticium rolfsii, ApplMicrobiol Biotechnol 50:323-330), Talaromyces glucoamylases, inparticular derived from Talaromyces emersonii (WO 99/28448), Talaromycesleycettanus (U.S. Pat. No. Re. 32,153), Talaromyces duponti, Talaromycesthermophilus (U.S. Pat. No. 4,587,215). In a preferred embodiment theglucoamylase used during saccharification and/or fermentation is theTalaromyces emersonii glucoamylase disclosed in WO 99/28448.

Bacterial glucoamylases contemplated include glucoamylases from thegenus Clostridium, in particular C. thermoamylolyticum (EP 135,138), andC. thermohydrosulfuricum (WO 86/01831).

Contemplated fungal glucoamylases include Trametes cingulate (SEQ ID NO:20), Pachykytospora papyracea; and Leucopaxillus giganteus all disclosedin WO 2006/069289; or Peniophora rufomarginata disclosed inWO2007/124285; or a mixture thereof. Also hybrid glucoamylase arecontemplated according to the invention. Examples include the hybridglucoamylases disclosed in WO 2005/045018. Specific examples include thehybrid glucoamylase disclosed in Table 1 and 4 of Example 1 (whichhybrids are hereby incorporated by reference).

In an embodiment the glucoamylase is derived from a strain of the genusPycnoporus, in particular a strain of Pycnoporus sanguineus as describedin WO 2011/066576 (SEQ ID NOs 2, 4 or 6), in particular the one shown aSEQ ID NO: 28 herein (corresponding to SEQ ID NO: 4 in WO 2011/066576)or from a strain of the genus Gloeophyllum, such as a strain ofGloeophyllum sepiarium or Gloeophyllum trabeum, in particular a strainof Gloeophyllum as described in WO 2011/068803 (SEQ ID NO: 2, 4, 6, 8,10, 12, 14 or 16). In a preferred embodiment the glucoamylase is SEQ IDNO: 2 in WO 2011/068803 or SEQ ID NO: 19 herein (i.e. Gloeophyllumsepiarium glucoamylase). In a preferred embodiment the glucoamylase isSEQ ID NO: 20 herein (i.e., Gloeophyllum trabeum glucoamylase disclosesas SEQ ID NO: 3 in WO2014/177546) (all references hereby incorporated byreference).

Contemplated are also glucoamylases which exhibit a high identity to anyof the above mentioned glucoamylases, i.e., at least 60%, such as atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% oreven 100% identity to any one of the mature parts of the enzymesequences mentioned above, such as any of SEQ ID NOs: 13, 14, 19, 20 or28 herein, respectively.

In an embodiment the glucoamylase used in fermentation or SSF exhibitsat least 60%, such as at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or even 100% identity to the mature part of SEQ ID NO:20 herein.

In an embodiment the glucoamylase used in fermentation or SSF exhibitsat least 60%, such as at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or even 100% identity to the mature part of SEQ ID NO:28 herein.

Glucoamylases may in an embodiment be added to the saccharificationand/or fermentation in an amount of 0.0001-20 AGU/g DS, preferably0.001-10 AGU/g DS, especially between 0.01-5 AGU/g DS, such as 0.1-2AGU/g DS.

Glucoamylases may in an embodiment be added to the saccharificationand/or fermentation in an amount of 1-1,000 μg EP/g DS, preferably10-500 μg/gDS, especially between 25-250 μg/g DS.

In an embodiment the glucoamylase is added as a blend further comprisingan alpha-amylase. In a preferred embodiment the alpha-amylase is afungal alpha-amylase, especially an acid fungal alpha-amylase. Thealpha-amylase is typically a side activity.

In an embodiment the glucoamylase is a blend comprising Talaromycesemersonii glucoamylase disclosed in WO 99/28448 as SEQ ID NO: 34 or SEQID NO: 13 herein and Trametes cingulata glucoamylase disclosed as SEQ IDNO: 2 in WO 06/069289 and SEQ ID NO: 14 herein.

In an embodiment the glucoamylase is a blend comprising Talaromycesemersonii glucoamylase disclosed in SEQ ID NO: 13 herein, Trametescingulata glucoamylase disclosed as SEQ ID NO: 14 herein, and Rhizomucorpusillus alpha-amylase with Aspergillus niger glucoamylase linker andSBD disclosed as V039 in Table 5 in WO 2006/069290 or SEQ ID NO: 15herein.

In an embodiment the glucoamylase is a blend comprising Gloeophyllumsepiarium glucoamylase shown as SEQ ID NO: 19 herein and Rhizomucorpusillus with an Aspergillus niger glucoamylase linker andstarch-binding domain (SBD), disclosed SEQ ID NO: 15 herein with thefollowing substitutions: G128D+D143N.

In an embodiment the alpha-amylase may be derived from a strain of thegenus Rhizomucor, preferably a strain the Rhizomucor pusillus, such asthe one shown in SEQ ID NO: 3 in WO2013/006756, or the genus Meripilus,preferably a strain of Meripilus giganteus. In a preferred embodimentthe alpha-amylase is derived from a Rhizomucor pusillus with anAspergillus niger glucoamylase linker and starch-binding domain (SBD),disclosed as V039 in Table 5 in WO 2006/069290 or SEQ ID NO: 15 herein.

In an embodiment the Rhizomucor pusillus alpha-amylase or the Rhizomucorpusillus alpha-amylase with a linker and starch-binding domain (SBD),preferably Aspergillus niger glucoamylase linker and SBD, has at leastone of the following substitutions or combinations of substitutions:D165M; Y141W; Y141R; K136F; K192R; P224A; P224R; S123H+Y141W;G20S+Y141W; A76G+Y141W; G128D+Y141W; G128D+D143N; P219C+Y141W;N142D+D143N; Y141W+K192R; Y141W+D143N; Y141W+N383R; Y141W+P219C+A265C;Y141W+N142D+D143N; Y141W+K192R V410A; G128D+Y141W+D143N;Y141W+D143N+P219C; Y141W+D143N+K192R; G128D+D143N+K192R;Y141W+D143N+K192R+P219C; G128D+Y141W+D143N+K192R; orG128D+Y141W+D143N+K192R+P219C (using SEQ ID NO: 3 in WO 2013/006756 fornumbering or SEQ ID NO: 15 herein).

In a preferred embodiment the glucoamylase blend comprises Gloeophyllumsepiarium glucoamylase (e.g., SEQ ID NO: 2 in WO 2011/068803 or SEQ IDNO: 19 herein) and Rhizomucor pusillus alpha-amylase.

In a preferred embodiment the glucoamylase blend comprises Gloeophyllumsepiarium glucoamylase shown as SEQ ID NO: 2 in WO 2011/068803 or SEQ IDNO: 19 herein and Rhizomucor pusillus with a linker and starch-bindingdomain (SBD), preferably Aspergillus niger glucoamylase linker and SBD,disclosed SEQ ID NO: 3 in WO 2013/006756 and SEQ ID NO: 15 herein withthe following substitutions: G128D+D143N.

Commercially available compositions comprising glucoamylase include AMG200L; AMG 300 L; SAN™ SUPER, SAN™ EXTRA L, SPIRIZYME™ PLUS, SPIRIZYME™FUEL, SPIRIZYME™ B4U, SPIRIZYME™ ULTRA, SPIRIZYME™ EXCEL, SPIRIZYMEACHIEVE™, and AMG™ E (from Novozymes A/S); OPTIDEX™ 300, GC480, GC417(from DuPont-Danisco); AMIGASE™ and AMIGASE™ PLUS (from DSM); G-ZYME™G900, G-ZYME™ and G990 ZR (from DuPont-Danisco).

Cellulolytic Enzyme Composition Present and/or Added in Saccharificationand/or Fermentation

According to the invention a cellulolytic enzyme composition may bepresent in saccharification, fermentation or simultaneoussaccharification and fermentation (SSF).

The cellulolytic enzyme composition comprises a beta-glucosidase, acellobiohydrolase and an endoglucanase.

Examples of suitable cellulolytic composition can be found in WO2008/151079 and WO 2013/028928 which are incorporated by reference.

In preferred embodiments the cellulolytic enzyme composition is derivedfrom a strain of Trichoderma, Humicola, or Chrysosporium.

In an embodiment the cellulolytic enzyme composition is derived from astrain of Trichoderma reesei, Humicola insolens and/or Chrysosporiumlucknowense.

In an embodiment the cellulolytic enzyme composition comprises abeta-glucosidase, preferably one derived from a strain of the genusAspergillus, such as Aspergillus oryzae, such as the one disclosed in WO2002/095014 or the fusion protein having beta-glucosidase activitydisclosed in WO 2008/057637 (in particular the Aspergillus oryzaebeta-glucosidase variant fusion protein shown in SEQ ID NOs: 73 and 74,respectively, in WO 2008/057637 or the Aspergillus oryzaebeta-glucosidase fusion protein shown in SEQ ID NOs: 75 and 76,respectively, in WO 2008/057637—both hereby incorporated by reference),or Aspergillus fumigatus, such as one disclosed in WO 2005/047499 or SEQID NO: 10 herein or an Aspergillus fumigatus beta-glucosidase variantdisclosed in WO 2012/044915 (Novozymes), such as one with one or more,such as all, of the following substitutions: F100D, S283G, N456E, F512Y;or a strain of the genus a strain Penicillium, such as a strain of thePenicillium brasilianum disclosed in WO 2007/019442, or a strain of thegenus Trichoderma, such as a strain of Trichoderma reesei.

In an embodiment the cellulolytic enzyme composition comprises a GH61polypeptide having cellulolytic enhancing activity such as one derivedfrom the genus Thermoascus, such as a strain of Thermoascus aurantiacus,such as the one described in WO 2005/074656 as SEQ ID NO: 2 or SEQ IDNO: 21 herein; or one derived from the genus Thielavia, such as a strainof Thielavia terrestris, such as the one described in WO 2005/074647 asSEQ ID NO: 7 and SEQ ID NO: 8; or one derived from a strain ofAspergillus, such as a strain of Aspergillus fumigatus, such as the onedescribed in WO 2010/138754 as SEQ ID NO: 1 and SEQ ID NO: 2; or onederived from a strain derived from Penicillium, such as a strain ofPenicillium emersonii, such as the one disclosed in WO 2011/041397 asSEQ ID NO: 2 or SEQ ID NO: 12 herein.

In an embodiment the cellulolytic composition comprises acellobiohydrolase I (CBH I), such as one derived from a strain of thegenus Aspergillus, such as a strain of Aspergillus fumigatus, such asthe Cel7a CBH I disclosed in SEQ ID NO: 2 in WO 2011/057140 or SEQ IDNO: 6 herein, or a strain of the genus Trichoderma, such as a strain ofTrichoderma reesei.

In an embodiment the cellulolytic composition comprises acellobiohydrolase II (CBH II, such as one derived from a strain of thegenus Aspergillus, such as a strain of Aspergillus fumigatus disclosedas SEQ ID NO: 8 herein; or a strain of the genus Trichoderma, such asTrichoderma reesei, or a strain of the genus Thielavia, such as a strainof Thielavia terrestris, such as cellobiohydrolase II CEL6A fromThielavia terrestris.

In an embodiment the cellulolytic enzyme composition comprises a GH61polypeptide having cellulolytic enhancing activity and abeta-glucosidase.

In an embodiment the cellulolytic composition comprises a GH61polypeptide having cellulolytic enhancing activity, a beta-glucosidase,and a CBH I.

In an embodiment the cellulolytic composition comprises a GH61polypeptide having cellulolytic enhancing activity, a beta-glucosidase,a CBH I, and a CBH II.

In an embodiment the cellulolytic enzyme composition is a Trichodermareesei cellulolytic enzyme composition, further comprising Thermoascusaurantiacus GH61A polypeptide having cellulolytic enhancing activity(SEQ ID NO: 2 in WO 2005/074656 or SEQ ID NO: 21 herein), andAspergillus oryzae beta-glucosidase fusion protein (WO 2008/057637).

In an embodiment the cellulolytic composition is a Trichoderma reeseicellulolytic enzyme composition, further comprising Thermoascusaurantiacus GH61A polypeptide having cellulolytic enhancing activity(SEQ ID NO: 2 in WO 2005/074656 or SEQ ID NO: 21 herein) and Aspergillusfumigatus beta-glucosidase (SEQ ID NO: 2 of WO 2005/047499 or SEQ ID NO:10 herein).

In an embodiment the cellulolytic enzyme composition is a Trichodermareesei cellulolytic enzyme composition further comprising Penicilliumemersonii GH61A polypeptide having cellulolytic enhancing activitydisclosed as SEQ ID NO: 2 in WO 2011/041397 or SEQ ID NO: 12 herein andAspergillus fumigatus beta-glucosidase (SEQ ID NO: 2 of WO 2005/047499or SEQ ID NO: 10 herein) or a variant thereof with the followingsubstitutions F100D, S283G, N456E, F512Y (using SEQ ID NO: 10 herein fornumbering).

In a preferred embodiment the cellulolytic enzyme composition comprisingone or more of the following components:

-   -   (i) an Aspergillus fumigatus cellobiohydrolase I;    -   (ii) an Aspergillus fumigatus cellobiohydrolase II;    -   (iii) an Aspergillus fumigatus beta-glucosidase or variant        thereof; and    -   (iv) a Penicillium sp. GH61 polypeptide having cellulolytic        enhancing activity; or homologs thereof.

In an preferred embodiment the cellulolytic enzyme composition isderived from Trichoderma reesei comprising GH61A polypeptide havingcellulolytic enhancing activity derived from a strain of Penicilliumemersonii (SEQ ID NO: 2 in WO 2011/041397 or SEQ ID NO: 12 herein),Aspergillus fumigatus beta-glucosidase (SEQ ID NO: 2 in WO 2005/047499or SEQ ID NO: 10 herein) variant with the following substitutions:F100D, S283G, N456E, F512Y (disclosed in WO 2012/044915); Aspergillusfumigatus Cel7A CBH I disclosed as SEQ ID NO: 6 in WO2011/057140 or SEQID NO: 6 herein and Aspergillus fumigatus CBH II disclosed as SEQ ID NO:18 in WO 2011/057140 or SEQ ID NO: 8 herein.

In an embodiment the cellulolytic composition is dosed from 0.0001-3 mgEP/g DS, preferably, 0.0005-2 mg EP/g DS, preferably 0.001-1 mg/g DS,more preferably 0.005-0.5 mg EP/g DS, and even more preferably 0.01-0.1mg EP/g DS.

Proteases Present and/or Added in Saccharification and/or Fermentation

Any suitable protease may be added in saccharification and/orfermentation, such as SSF.

In a preferred embodiment the protease is a metallo protease or a serineprotease.

In an embodiment the enzyme composition comprises a metallo protease,preferably derived from a strain of the genus Thermoascus, preferably astrain of Thermoascus aurantiacus, especially Thermoascus aurantiacusCGMCC No. 0670, such as the metallo protease disclosed as the maturepart of SEQ ID NO: 2 disclosed in WO 2003/048353 or the maturepolypeptide of SEQ ID NO: 17 herein.

In an embodiment the protease has at least 60%, such as at least 70%,such as at least 75% identity preferably at least 80%, more preferablyat least 85%, more preferably at least 90%, more preferably at least91%, more preferably at least 92%, even more preferably at least 93%,most preferably at least 94%, and even most preferably at least 95%,such as even at least 96%, at least 97%, at least 98%, at least 99%, or100% identity to the mature part of the polypeptide of SEQ ID NO: 17herein.

In an embodiment the protease is derived from a strain of Pyrococcus,such as a strain of Pyrococcus furiosus, such as the protease shown inSEQ ID NO: 1 in U.S. Pat. No. 6,358,726 or SEQ ID NO: 22 herein.

In an embodiment the protease is the mature sequence from Meripilusgiganteus protease 3 (peptidase family S53 protease) concerned inExample 2 in WO 2014/037438 (hereby incorporated by reference). In anembodiment the protease is the mature protease 3 sequence from a strainof Meripilus, in particular Meripilus giganteus shown as SEQ ID NO: 5 inWO 2014/037438 (hereby incorporated by reference) and SEQ ID NO: 32herein.

In an embodiment the protease has at least 60%, such as at least 70%,such as at least 75% identity preferably at least 80%, more preferablyat least 85%, more preferably at least 90%, more preferably at least91%, more preferably at least 92%, even more preferably at least 93%,most preferably at least 94%, and even most preferably at least 95%,such as even at least 96%, at least 97%, at least 98%, at least 99%, or100% identity to the mature part of the polypeptide of SEQ ID NO: 32herein shown as amino acids 1-547.

Alpha-Amylase Present and/or Added in Saccharification and/orFermentation

Any suitable alpha-amylase, such as fungal acid alpha-amylase, may bepresent and/or added in saccharification and/or fermentation.

In a preferably embodiment the alpha-amylase is a fungal alpha-amylase,in particular one that has at least 60%, such as at least 70%, such asat least 75% identity preferably at least 80%, more preferably at least85%, more preferably at least 90%, more preferably at least 91%, morepreferably at least 92%, even more preferably at least 93%, mostpreferably at least 94%, and even most preferably at least 95%, such aseven at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to the mature part of the polypeptide of SEQ ID NO: 15. In apreferred embodiment the alpha-amylase has one or more of the followingsubstitutions: G128D, D143N, in particular G128D+D143N.

Processes of Producing a Fermentation Product From Cellulolic MaterialsUsing a Trehalase of the Invention

In an embodiment the invention relates to processes of producing afermentation product from pretreated cellulosic material, comprising:

-   -   (a) hydrolyzing said pretreated cellulosic material with a        cellulolytic enzyme composition;    -   (b) fermenting using a fermenting organism; and    -   (c) optionally recovering the fermentation product,    -   wherein a trehalase of the invention is added and/or present in        hydrolysis step (a) and/or fermentation step (b).

According to the process of the invention hydrolysis and fermentationmay be carried out separate or simultaneous. In an embodiment theprocess of the invention is carried out as separate hydrolysis andfermentation (SHF); simultaneous saccharification and fermentation(SSF); simultaneous saccharification and co-fermentation (SSCF); hybridhydrolysis and fermentation (HHF); separate hydrolysis andco-fermentation (SHCF); hybrid hydrolysis and co-fermentation (HHCF); ordirect microbial conversion (DMC), also sometimes called consolidatedbioprocessing (CBP). SHF uses separate process steps to firstenzymatically hydrolyze the cellulosic material to fermentable sugars,e.g., glucose, cellobiose, and pentose monomers, and then ferment thefermentable sugars to ethanol. In SSF, the enzymatic hydrolysis of thecellulosic material and the fermentation of sugars to ethanol arecombined in one step. SSCF involves the co-fermentation of multiplesugars (Sheehan, J., and Himmel, M., 1999, Enzymes, energy and theenvironment: A strategic perspective on the U.S. Department of Energy'sresearch and development activities for bioethanol, Biotechnol. Prog.15: 817-827). HHF involves a separate hydrolysis step, and in addition asimultaneous saccharification and hydrolysis step, which can be carriedout in the same reactor. The steps in a HHF process can be carried outat different temperatures, i.e., high temperature enzymatic hydrolysisfollowed by SSF at a lower temperature that the fermentation strain cantolerate. DMC combines all three processes (enzyme production,hydrolysis, and fermentation) in one or more (e.g., several) steps wherethe same organism is used to produce the enzymes for conversion of thecellulosic material to fermentable sugars and to convert the fermentablesugars into a final product.

According to the invention the cellulosic material is plant materialchips, plant stem segments and/or whole plant stems. In an embodimentcellulosic material is selected from the group comprising arundo,bagasse, bamboo, corn cob, corn fiber, corn stover, miscanthus, orangepeel, rice straw, switchgrass, wheat straw. In a preferred embodimentthe source of the cellulosic material is corn stover, corn cobs, and/orwheat straw.

According to the invention any pretreatment may be used. In a preferredembodiment chemical pretreatment, physical pretreatment, or chemicalpretreatment and a physical pretreatment is used. In a preferredembodiment the cellulosic material is pretreated with an acid, such asdilute acid pretreatment. In an embodiment the cellulosic material isprepared by pretreating cellulosic material at high temperature, highpressure with an acid.

In an embodiment hydrolysis is carried out at a temperature between20-70° C., such as 30-60° C., preferably 45-55° C. at a pH in the range4-6, such as 4.5-5.5.

In an embodiment the cellulosic material is present at 1-20 (w/w) % ofTS, such as 2-10 (w/w) % TS, such as around 5 (w/w) % TS duringhydrolysis.

In an embodiment the hydrolysis is carried out for 1-20 days, preferablybetween from 5-15 days.

In an embodiment the cellulolytic enzyme composition is derived fromTrichoderma reesei, Humicola insolens or Chrysosporium lucknowense.

Cellulolytic enzyme composition: The term “cellulolytic enzymecomposition” means one or more (e.g., several) enzymes that hydrolyze acellulosic material. Such enzymes include endoglucanase(s),cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. Thetwo basic approaches for measuring cellulolytic activity include: (1)measuring the total cellulolytic activity, and (2) measuring theindividual cellulolytic activities (endoglucanases, cellobiohydrolases,and beta-glucosidases) as reviewed in Zhang et al., Outlook forcellulase improvement: Screening and selection strategies, 2006,Biotechnology Advances 24: 452-481. Total cellulolytic activity isusually measured using insoluble substrates, including Whatman No 1filter paper, microcrystalline cellulose, bacterial cellulose, algalcellulose, cotton, pretreated lignocellulose, etc. The most common totalcellulolytic activity assay is the filter paper assay using Whatman No 1filter paper as the substrate. The assay was established by theInternational Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987,Measurement of cellulase activities, Pure Appl. Chem. 59: 257-68).

For purposes of the present invention, cellulolytic enzyme activity isdetermined by measuring the increase in hydrolysis of a cellulosicmaterial by cellulolytic enzyme(s) under the following conditions: 1-50mg of cellulolytic enzyme protein/g of cellulose in PCS (or otherpretreated cellulosic material) for 3-7 days at a suitable temperature,e.g., 50° C., 55° C., or 60° C., compared to a control hydrolysiswithout addition of cellulolytic enzyme protein. Typical conditions are1 ml reactions, washed or unwashed PCS, 5% insoluble solids, 50 mMsodium acetate pH 5, 1 mM MnSO₄, 50° C., 55° C., or 60° C., 72 hours,sugar analysis by AMINEX® HPX-87H column (Bio-Rad Laboratories, Inc.,Hercules, Calif., USA).

Examples of cellulolytic compositions can be found in the “CellulolyticEnzyme Composition present and/or added during Saccharification and/orFermentation”-section above.

Cellulosic material: The term “cellulosic material” means any materialcontaining cellulose. The predominant polysaccharide in the primary cellwall of cellulosic material is cellulose, the second most abundant ishemicellulose, and the third is pectin. The secondary cell wall,produced after the cell has stopped growing, also containspolysaccharides and is strengthened by polymeric lignin covalentlycross-linked to hemicellulose. Cellulose is a homopolymer ofanhydrocellobiose and thus a linear beta-(1-4)-D-glucan, whilehemicelluloses include a variety of compounds, such as xylans,xyloglucans, arabinoxylans, and mannans in complex branched structureswith a spectrum of substituents. Although generally polymorphous,cellulose is found in plant tissue primarily as an insoluble crystallinematrix of parallel glucan chains. Hemicelluloses usually hydrogen bondto cellulose, as well as to other hemicelluloses, which help stabilizethe cell wall matrix.

Cellulose is generally found, for example, in the stems, leaves, hulls,husks, and cobs of plants or leaves, branches, and wood of trees. Thecellulosic material can be, but is not limited to, agricultural residue,herbaceous material (including energy crops), municipal solid waste,pulp and paper mill residue, waste paper, and wood (including forestryresidue) (see, for example, Wiselogel et al., 1995, in Handbook onBioethanol (Charles E. Wyman, editor), pp. 105-118, Taylor & Francis,Washington D.C.; Wyman, 1994, Bioresource Technology 50: 3-16; Lynd,1990, Applied Biochemistry and Biotechnology 24/25: 695-719; Mosier etal., 1999, Recent Progress in Bioconversion of Lignocellulosics, inAdvances in Biochemical Engineering/Biotechnology, T. Scheper, managingeditor, Volume 65, pp. 23-40, Springer-Verlag, N.Y.). It is understoodherein that the cellulose may be in the form of lignocellulose, a plantcell wall material containing lignin, cellulose, and hemicellulose in amixed matrix. In a preferred aspect, the cellulosic material is anybiomass material. In another preferred aspect, the cellulosic materialis lignocellulose, which comprises cellulose, hemicelluloses, andlignin.

In one aspect, the cellulosic material is agricultural residue. Inanother aspect, the cellulosic material is herbaceous material(including energy crops). In another aspect, the cellulosic material ismunicipal solid waste. In another aspect, the cellulosic material ispulp and paper mill residue. In another aspect, the cellulosic materialis waste paper. In another aspect, the cellulosic material is wood(including forestry residue).

In another aspect, the cellulosic material is arundo. In another aspect,the cellulosic material is bagasse. In another aspect, the cellulosicmaterial is bamboo. In another aspect, the cellulosic material is corncob. In another aspect, the cellulosic material is corn fiber. Inanother aspect, the cellulosic material is corn stover. In anotheraspect, the cellulosic material is miscanthus. In another aspect, thecellulosic material is orange peel. In another aspect, the cellulosicmaterial is rice straw. In another aspect, the cellulosic material isswitchgrass. In another aspect, the cellulosic material is wheat straw.

In another aspect, the cellulosic material is aspen. In another aspect,the cellulosic material is eucalyptus. In another aspect, the cellulosicmaterial is fir. In another aspect, the cellulosic material is pine. Inanother aspect, the cellulosic material is poplar. In another aspect,the cellulosic material is spruce. In another aspect, the cellulosicmaterial is willow.

In another aspect, the cellulosic material is algal cellulose. Inanother aspect, the cellulosic material is bacterial cellulose. Inanother aspect, the cellulosic material is cotton linter. In anotheraspect, the cellulosic material is filter paper. In another aspect, thecellulosic material is microcrystalline cellulose. In another aspect,the cellulosic material is phosphoric-acid treated cellulose.

In another aspect, the cellulosic material is an aquatic biomass. Asused herein the term “aquatic biomass” means biomass produced in anaquatic environment by a photosynthesis process. The aquatic biomass canbe algae, emergent plants, floating-leaf plants, or submerged plants.

The cellulosic material may be used as is or may be subjected topretreatment, using conventional methods known in the art, as describedherein. In a preferred aspect, the cellulosic material is pretreated.

Beta-glucosidase: The term “beta-glucosidase” means a beta-D-glucosideglucohydrolase (E.C. 3.2.1.21) that catalyzes the hydrolysis of terminalnon-reducing beta-D-glucose residues with the release of beta-D-glucose.For purposes of the present invention, beta-glucosidase activity isdetermined using p-nitrophenyl-beta-D-glucopyranoside as substrateaccording to the procedure of Venturi et al., 2002, Extracellularbeta-D-glucosidase from Chaetomium thermophilum var. coprophilum:production, purification and some biochemical properties, J. BasicMicrobiol. 42: 55-66. One unit of beta-glucosidase is defined as 1.0μmole of p-nitrophenolate anion produced per minute at 25° C., pH 4.8from 1 mM p-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mMsodium citrate containing 0.01% TWEEN® 20 (polyoxyethylene sorbitanmonolaurate).

Beta-xylosidase: The term “beta-xylosidase” means a beta-D-xylosidexylohydrolase (E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of shortbeta (1→4)-xylooligosaccharides to remove successive D-xylose residuesfrom non-reducing termini. For purposes of the present invention, oneunit of beta-xylosidase is defined as 1.0 μmole of p-nitrophenolateanion produced per minute at 40° C., pH 5 from 1 mMp-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodium citratecontaining 0.01% TWEEN® 20.

Cellobiohydrolase: The term “cellobiohydrolase” means a1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91) that catalyzes thehydrolysis of 1,4-beta-D-glucosidic linkages in cellulose,cellooligosaccharides, or any beta-1,4-linked glucose containingpolymer, releasing cellobiose from the reducing or non-reducing ends ofthe chain (Teeri, 1997, Crystalline cellulose degradation: New insightinto the function of cellobiohydrolases, Trends in Biotechnology 15:160-167; Teeri et al., 1998, Trichoderma reesei cellobiohydrolases: whyso efficient on crystalline cellulose?, Biochem. Soc. Trans. 26:173-178). Cellobiohydrolase activity is determined according to theprocedures described by Lever et al., 1972, Anal. Biochem. 47: 273-279;van Tilbeurgh et al., 1982, FEBS Letters 149: 152-156; van Tilbeurgh andClaeyssens, 1985, FEBS Letters 187: 283-288; and Tomme et al., 1988,Eur. J. Biochem. 170: 575-581. In the present invention, the Tomme etal. method can be used to determine cellobiohydrolase activity.

Endoglucanase: The term “endoglucanase” means anendo-1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4) thatcatalyzes endohydrolysis of 1,4-beta-D-glycosidic linkages in cellulose,cellulose derivatives (such as carboxymethyl cellulose and hydroxyethylcellulose), lichenin, beta-1,4 bonds in mixed beta-1,3 glucans such ascereal beta-D-glucans or xyloglucans, and other plant materialcontaining cellulosic components. Endoglucanase activity can bedetermined by measuring reduction in substrate viscosity or increase inreducing ends determined by a reducing sugar assay (Zhang et al., 2006,Biotechnology Advances 24: 452-481). For purposes of the presentinvention, endoglucanase activity is determined using carboxymethylcellulose (CMC) as substrate according to the procedure of Ghose, 1987,Pure and Appl. Chem. 59: 257-268, at pH 5, 40° C.

Family 61 glycoside hydrolase: The term “Family 61 glycoside hydrolase”or “Family GH61” or “GH61” means a polypeptide falling into theglycoside hydrolase Family 61 according to Henrissat B., 1991, Aclassification of glycosyl hydrolases based on amino-acid sequencesimilarities, Biochem. J. 280: 309-316, and Henrissat and Bairoch, 1996,Updating the sequence-based classification of glycosyl hydrolases,Biochem. J. 316: 695-696. The enzymes in this family were originallyclassified as a glycoside hydrolase family based on measurement of veryweak endo-1,4-beta-D-glucanase activity in one family member. Thestructure and mode of action of these enzymes are non-canonical and theycannot be considered as bona fide glycosidases. However, they are keptin the CAZy classification on the basis of their capacity to enhance thebreakdown of cellulose when used in conjunction with a cellulase or amixture of cellulases.

Hemicellulolytic enzyme or hemicellulase: The term “hemicellulolyticenzyme” or “hemicellulase” means one or more (e.g., several) enzymesthat hydrolyze a hemicellulosic material. See, for example, Shallom andShoham, 2003, Microbial hemicellulases. Current Opinion In Microbiology6(3): 219-228). Hemicellulases are key components in the degradation ofplant biomass. Examples of hemicellulases include, but are not limitedto, an acetylmannan esterase, an acetylxylan esterase, an arabinanase,an arabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, agalactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, amannosidase, a xylanase, and a xylosidase. The substrates of theseenzymes, the hemicelluloses, are a heterogeneous group of branched andlinear polysaccharides that are bound via hydrogen bonds to thecellulose microfibrils in the plant cell wall, crosslinking them into arobust network. Hemicelluloses are also covalently attached to lignin,forming together with cellulose a highly complex structure. The variablestructure and organization of hemicelluloses require the concertedaction of many enzymes for its complete degradation. The catalyticmodules of hemicellulases are either glycoside hydrolases (GHs) thathydrolyze glycosidic bonds, or carbohydrate esterases (CEs), whichhydrolyze ester linkages of acetate or ferulic acid side groups. Thesecatalytic modules, based on homology of their primary sequence, can beassigned into GH and CE families. Some families, with an overall similarfold, can be further grouped into clans, marked alphabetically (e.g.,GH-A). A most informative and updated classification of these and othercarbohydrate active enzymes is available in the Carbohydrate-ActiveEnzymes (CAZy) database. Hemicellulolytic enzyme activities can bemeasured according to Ghose and Bisaria, 1987, Pure & Appl. Chem. 59:1739-1752, at a suitable temperature, e.g., 50° C., 55° C., or 60° C.,and pH, e.g., 5.0 or 5.5.

Polypeptide having cellulolytic enhancing activity: The term“polypeptide having cellulolytic enhancing activity” means a GH61polypeptide that catalyzes the enhancement of the hydrolysis of acellulosic material by enzyme having cellulolytic activity. For purposesof the present invention, cellulolytic enhancing activity is determinedby measuring the increase in reducing sugars or the increase of thetotal of cellobiose and glucose from the hydrolysis of a cellulosicmaterial by cellulolytic enzyme under the following conditions: 1-50 mgof total protein/g of cellulose in PCS, wherein total protein iscomprised of 50-99.5% w/w cellulolytic enzyme protein and 0.5-50% w/wprotein of a GH61 polypeptide having cellulolytic enhancing activity for1-7 days at a suitable temperature, e.g., 50° C., 55° C., or 60° C., andpH, e.g., 5.0 or 5.5, compared to a control hydrolysis with equal totalprotein loading without cellulolytic enhancing activity (1-50 mg ofcellulolytic protein/g of cellulose in PCS). In an aspect, a mixture ofCELLUCLAST® 1.5 L (Novozymes A/S, Bagsværd, Denmark) in the presence of2-3% of total protein weight Aspergillus oryzae beta-glucosidase(recombinantly produced in Aspergillus oryzae according to WO 02/095014)or 2-3% of total protein weight Aspergillus fumigatus beta-glucosidase(recombinantly produced in Aspergillus oryzae as described in WO2002/095014) of cellulase protein loading is used as the source of thecellulolytic activity.

The GH61 polypeptides having cellulolytic enhancing activity enhance thehydrolysis of a cellulosic material catalyzed by enzyme havingcellulolytic activity by reducing the amount of cellulolytic enzymerequired to reach the same degree of hydrolysis preferably at least1.01-fold, e.g., at least 1.05-fold, at least 1.10-fold, at least1.25-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least4-fold, at least 5-fold, at least 10-fold, or at least 20-fold.

Xylanase: The term “xylanase” means a 1,4-beta-D-xylan-xylohydrolase(E.C. 3.2.1.8) that catalyzes the endohydrolysis of 1,4-beta-D-xylosidiclinkages in xylans. For purposes of the present invention, xylanaseactivity is determined with 0.2% AZCL-arabinoxylan as substrate in 0.01%TRITON® X-100 and 200 mM sodium phosphate buffer pH 6 at 37° C. One unitof xylanase activity is defined as 1.0 μmole of azurine produced perminute at 37° C., pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200mM sodium phosphate pH 6 buffer.

Fermenting Organism for Cellulosic Based Fermentation

The term “fermenting organism” or ‘fermenting microorganism” refers toany organism, including bacterial and fungal organisms, suitable for usein a desired fermentation process to produce a fermentation product. Thefermenting organism may be hexose and/or pentose fermenting organisms,or a combination thereof. Both hexose and pentose fermenting organismsare well known in the art. Suitable fermenting microorganisms are ableto ferment, i.e., convert, sugars, such as glucose, xylose, xylulose,arabinose, maltose, mannose, galactose, and/or oligosaccharides,directly or indirectly into the desired fermentation product. Examplesof bacterial and fungal fermenting organisms producing ethanol aredescribed by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69: 627-642.

Examples of fermenting organisms that can ferment hexose sugars includebacterial and fungal organisms, such as yeast. Preferred yeast includesstrains of Candida, Kluyveromyces, and Saccharomyces, e.g., Candidasonorensis, Kluyveromyces marxianus, and Saccharomyces cerevisiae.

Examples of fermenting organisms that can ferment pentose sugars intheir native state include bacterial and fungal organisms, such as someyeast. Preferred xylose fermenting yeast include strains of Candida,preferably C. sheatae or C. sonorensis; and strains of Pichia,preferably P. stipitis, such as P. stipitis CBS 5773. Preferred pentosefermenting yeast include strains of Pachysolen, preferably P.tannophilus. Organisms not capable of fermenting pentose sugars, such asxylose and arabinose, may be genetically modified to do so by methodsknown in the art.

Examples of bacteria that can efficiently ferment hexose and pentose toethanol include, for example, Bacillus coagulans, Clostridiumacetobutylicum, Clostridium thermocellum, Clostridium phytofermentans,Geobacillus sp., Thermoanaerobacter saccharolyticum, and Zymomonasmobilis (Philippidis, 1996, supra).

Other fermenting organisms include strains of Bacillus, such as Bacilluscoagulans; Candida, such as C. sonorensis, C. methanosorbosa, C.diddensiae, C. parapsilosis, C. naedodendra, C. blankii, C.entomophilia, C. brassicae, C. pseudotropicalis, C. boidinii, C. utilis,and C. scehatae; Clostridium, such as C. acetobutylicum, C.thermocellum, and C. phytofermentans; E. coli, especially E. colistrains that have been genetically modified to improve the yield ofethanol; Geobacillus sp.; Hansenula, such as Hansenula anomala;Klebsiella, such as K. oxytoca; Kluyveromyces, such as K. marxianus, K.lactis, K. thermotolerans, and K. fragilis; Schizosaccharomyces, such asS. pombe; Thermoanaerobacter, such as Thermoanaerobactersaccharolyticum; and Zymomonas, such as Zymomonas mobilis.

In a preferred aspect, the yeast is a Bretannomyces. In a more preferredaspect, the yeast is Bretannomyces clausenii. In another preferredaspect, the yeast is a Candida. In another more preferred aspect, theyeast is Candida sonorensis. In another more preferred aspect, the yeastis Candida boidinii. In another more preferred aspect, the yeast isCandida blankii. In another more preferred aspect, the yeast is Candidabrassicae. In another more preferred aspect, the yeast is Candidadiddensii. In another more preferred aspect, the yeast is Candidaentomophiliia. In another more preferred aspect, the yeast is Candidapseudotropicalis. In another more preferred aspect, the yeast is Candidascehatae. In another more preferred aspect, the yeast is Candida utilis.In another preferred aspect, the yeast is a Clavispora. In another morepreferred aspect, the yeast is Clavispora lusitaniae. In another morepreferred aspect, the yeast is Clavispora opuntiae. In another preferredaspect, the yeast is a Kluyveromyces. In another more preferred aspect,the yeast is Kluyveromyces fragilis. In another more preferred aspect,the yeast is Kluyveromyces marxianus. In another more preferred aspect,the yeast is Kluyveromyces thermotolerans. In another preferred aspect,the yeast is a Pachysolen. In another more preferred aspect, the yeastis Pachysolen tannophilus. In another preferred aspect, the yeast is aPichia. In another more preferred aspect, the yeast is a Pichiastipitis. In another preferred aspect, the yeast is a Saccharomyces spp.In a more preferred aspect, the yeast is Saccharomyces cerevisiae. Inanother more preferred aspect, the yeast is Saccharomyces distaticus. Inanother more preferred aspect, the yeast is Saccharomyces uvarum.

In a preferred aspect, the bacterium is a Bacillus. In a more preferredaspect, the bacterium is Bacillus coagulans. In another preferredaspect, the bacterium is a Clostridium. In another more preferredaspect, the bacterium is Clostridium acetobutylicum. In another morepreferred aspect, the bacterium is Clostridium phytofermentans. Inanother more preferred aspect, the bacterium is Clostridiumthermocellum. In another more preferred aspect, the bacterium isGeobacilus sp. In another more preferred aspect, the bacterium is aThermoanaerobacter. In another more preferred aspect, the bacterium isThermoanaerobacter saccharolyticum. In another preferred aspect, thebacterium is a Zymomonas. In another more preferred aspect, thebacterium is Zymomonas mobilis.

Commercially available yeast suitable for ethanol production include,e.g., BIOFERM™ AFT and XR (NABC—North American Bioproducts Corporation,GA, USA), ETHANOL RED™ yeast (Fermentis/Lesaffre, USA), FALI™(Fleischmann's Yeast, USA), FERMIOL™ (DSM Specialties), GERT STRAND™(Gert Strand AB, Sweden), and SUPERSTART™ and THERMOSACC™ fresh yeast(Ethanol Technology, WI, USA).

In a preferred aspect, the fermenting microorganism has been geneticallymodified to provide the ability to ferment pentose sugars, such asxylose utilizing, arabinose utilizing, and xylose and arabinoseco-utilizing microorganisms.

The cloning of heterologous genes into various fermenting microorganismshas led to the construction of organisms capable of converting hexosesand pentoses to ethanol (co-fermentation) (Chen and Ho, 1993, Cloningand improving the expression of Pichia stipitis xylose reductase gene inSaccharomyces cerevisiae, Appl. Biochem. Biotechnol. 39-40: 135-147; Hoet al., 1998, Genetically engineered Saccharomyces yeast capable ofeffectively cofermenting glucose and xylose, Appl. Environ. Microbiol.64: 1852-1859; Kotter and Ciriacy, 1993, Xylose fermentation bySaccharomyces cerevisiae, Appl. Microbiol. Biotechnol. 38: 776-783;Walfridsson et al., 1995, Xylose-metabolizing Saccharomyces cerevisiaestrains overexpressing the TKL1 and TAL1 genes encoding the pentosephosphate pathway enzymes transketolase and transaldolase, Appl.Environ. Microbiol. 61: 4184-4190; Kuyper et al., 2004, Minimalmetabolic engineering of Saccharomyces cerevisiae for efficientanaerobic xylose fermentation: a proof of principle, FEMS Yeast Research4: 655-664; Beall et al., 1991, Parametric studies of ethanol productionfrom xylose and other sugars by recombinant Escherichia coli, Biotech.Bioeng. 38: 296-303; Ingram et al., 1998, Metabolic engineering ofbacteria for ethanol production, Biotechnol. Bioeng. 58: 204-214; Zhanget al., 1995, Metabolic engineering of a pentose metabolism pathway inethanologenic Zymomonas mobilis, Science 267: 240-243; Deanda et al.,1996, Development of an arabinose-fermenting Zymomonas mobilis strain bymetabolic pathway engineering, Appl. Environ. Microbiol. 62: 4465-4470;WO 2003/062430, xylose isomerase).

In a preferred aspect, the genetically modified fermenting microorganismis Candida sonorensis. In another preferred aspect, the geneticallymodified fermenting microorganism is Escherichia coli. In anotherpreferred aspect, the genetically modified fermenting microorganism isKlebsiella oxytoca. In another preferred aspect, the geneticallymodified fermenting microorganism is Kluyveromyces marxianus. In anotherpreferred aspect, the genetically modified fermenting microorganism isSaccharomyces cerevisiae. In another preferred aspect, the geneticallymodified fermenting microorganism is Zymomonas mobilis.

It is well known in the art that the organisms described above can alsobe used to produce other substances, as described herein.

The fermenting microorganism is typically added to the degradedcellulosic material or hydrolysate and the fermentation is performed forabout 8 to about 96 hours, e.g., about 24 to about 60 hours. Thetemperature is typically between about 26° C. to about 60° C., e.g.,about 32° C. or 50° C., and about pH 3 to about pH 8, e.g., pH 4-5, 6,or 7.

In one aspect, the yeast and/or another microorganism are applied to thedegraded cellulosic material and the fermentation is performed for about12 to about 96 hours, such as typically 24-60 hours. In another aspect,the temperature is preferably between about 20° C. to about 60° C.,e.g., about 25° C. to about 50° C., about 32° C. to about 50° C., orabout 32° C. to about 50° C., and the pH is generally from about pH 3 toabout pH 7, e.g., about pH 4 to about pH 7. However, some fermentingorganisms, e.g., bacteria, have higher fermentation temperature optima.Yeast or another microorganism is preferably applied in amounts ofapproximately 10⁵ to 10¹², preferably from approximately 10⁷ to 10¹⁰,especially approximately 2×10⁸ viable cell count per ml of fermentationbroth. Further guidance in respect of using yeast for fermentation canbe found in, e.g., “The Alcohol Textbook” (Editors K. Jacques, T. P.Lyons and D. R. Kelsall, Nottingham University Press, United Kingdom1999), which is hereby incorporated by reference.

For ethanol production, following the fermentation the fermented slurryis distilled to extract the ethanol. The ethanol obtained according tothe processes of the invention can be used as, e.g., fuel ethanol,drinking ethanol, i.e., potable neutral spirits, or industrial ethanol.

A fermentation stimulator can be used in combination with any of theprocesses described herein to further improve the fermentation process,and in particular, the performance of the fermenting microorganism, suchas, rate enhancement and ethanol yield. A “fermentation stimulator”refers to stimulators for growth of the fermenting microorganisms, inparticular, yeast. Preferred fermentation stimulators for growth includevitamins and minerals. Examples of vitamins include multivitamins,biotin, pantothenate, nicotinic acid, meso-inositol, thiamine,pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and VitaminsA, B, C, D, and E. See, for example, Alfenore et al., Improving ethanolproduction and viability of Saccharomyces cerevisiae by a vitaminfeeding strategy during fed-batch process, Springer-Verlag (2002), whichis hereby incorporated by reference. Examples of minerals includeminerals and mineral salts that can supply nutrients comprising P, K,Mg, S, Ca, Fe, Zn, Mn, and Cu.

Fermentation Products

According to the invention the term “fermentation product” can be anysubstance derived from fermentation. The fermentation product can be,without limitation, an alcohol (e.g., arabinitol, n-butanol, isobutanol,ethanol, glycerol, methanol, ethylene glycol, 1,3-propanediol [propyleneglycol], butanediol, glycerin, sorbitol, and xylitol); an alkane (e.g.,pentane, hexane, heptane, octane, nonane, decane, undecane, anddodecane), a cycloalkane (e.g., cyclopentane, cyclohexane, cycloheptane,and cyclooctane), an alkene (e.g. pentene, hexene, heptene, and octene);an amino acid (e.g., aspartic acid, glutamic acid, glycine, lysine,serine, and threonine); a gas (e.g., methane, hydrogen (H₂), carbondioxide (CO₂), and carbon monoxide (CO)); isoprene; a ketone (e.g.,acetone); an organic acid (e.g., acetic acid, acetonic acid, adipicacid, ascorbic acid, citric acid, 2,5-diketo-D-gluconic acid, formicacid, fumaric acid, glucaric acid, gluconic acid, glucuronic acid,glutaric acid, 3-hydroxypropionic acid, itaconic acid, lactic acid,malic acid, malonic acid, oxalic acid, oxaloacetic acid, propionic acid,succinic acid, and xylonic acid); and polyketide. The fermentationproduct can also be protein as a high value product.

In a preferred aspect, the fermentation product is an alcohol. It willbe understood that the term “alcohol” encompasses a substance thatcontains one or more (e.g., several) hydroxyl moieties. In a morepreferred aspect, the alcohol is n-butanol. In another more preferredaspect, the alcohol is isobutanol. In another more preferred aspect, thealcohol is ethanol. In another more preferred aspect, the alcohol ismethanol. In another more preferred aspect, the alcohol is arabinitol.In another more preferred aspect, the alcohol is butanediol. In anothermore preferred aspect, the alcohol is ethylene glycol. In another morepreferred aspect, the alcohol is glycerin. In another more preferredaspect, the alcohol is glycerol. In another more preferred aspect, thealcohol is 1,3-propanediol. In another more preferred aspect, thealcohol is sorbitol. In another more preferred aspect, the alcohol isxylitol. See, for example, Gong, C. S., Cao, N. J., Du, J., and Tsao, G.T., 1999, Ethanol production from renewable resources, in Advances inBiochemical Engineering/Biotechnology, Scheper, T., ed., Springer-VerlagBerlin Heidelberg, Germany, 65: 207-241; Silveira, M. M., and Jonas, R.,2002, The biotechnological production of sorbitol, Appl. Microbiol.Biotechnol. 59: 400-408; Nigam and Singh, 1995, Processes forfermentative production of xylitol—a sugar substitute, ProcessBiochemistry 30(2): 117-124; Ezeji et al., 2003, Production of acetone,butanol and ethanol by Clostridium beijerinckii BA101 and in siturecovery by gas stripping, World Journal of Microbiology andBiotechnology 19(6): 595-603.

In another preferred aspect, the fermentation product is an alkane. Thealkane can be an unbranched or a branched alkane. In another morepreferred aspect, the alkane is pentane. In another more preferredaspect, the alkane is hexane. In another more preferred aspect, thealkane is heptane. In another more preferred aspect, the alkane isoctane. In another more preferred aspect, the alkane is nonane. Inanother more preferred aspect, the alkane is decane. In another morepreferred aspect, the alkane is undecane. In another more preferredaspect, the alkane is dodecane.

In another preferred aspect, the fermentation product is a cycloalkane.In another more preferred aspect, the cycloalkane is cyclopentane. Inanother more preferred aspect, the cycloalkane is cyclohexane. Inanother more preferred aspect, the cycloalkane is cycloheptane. Inanother more preferred aspect, the cycloalkane is cyclooctane.

In another preferred aspect, the fermentation product is an alkene. Thealkene can be an unbranched or a branched alkene. In another morepreferred aspect, the alkene is pentene. In another more preferredaspect, the alkene is hexene. In another more preferred aspect, thealkene is heptene. In another more preferred aspect, the alkene isoctene.

In another preferred aspect, the fermentation product is an amino acid.In another more preferred aspect, the organic acid is aspartic acid. Inanother more preferred aspect, the amino acid is glutamic acid. Inanother more preferred aspect, the amino acid is glycine. In anothermore preferred aspect, the amino acid is lysine. In another morepreferred aspect, the amino acid is serine. In another more preferredaspect, the amino acid is threonine. See, for example, Richard andMargaritis, 2004, Empirical modeling of batch fermentation kinetics forpoly(glutamic acid) production and other microbial biopolymers,Biotechnology and Bioengineering 87(4): 501-515.

In another preferred aspect, the fermentation product is a gas. Inanother more preferred aspect, the gas is methane. In another morepreferred aspect, the gas is H₂. In another more preferred aspect, thegas is CO₂. In another more preferred aspect, the gas is CO. See, forexample, Kataoka, Miya, and Kiriyama, 1997, Studies on hydrogenproduction by continuous culture system of hydrogen-producing anaerobicbacteria, Water Science and Technology 36(6-7): 41-47; and Gunaseelan,1997, Anaerobic digestion of biomass for methane production: A review,Biomass and Bioenergy, 13(1-2): 83-114.

In another preferred aspect, the fermentation product is isoprene.

In another preferred aspect, the fermentation product is a ketone. Itwill be understood that the term “ketone” encompasses a substance thatcontains one or more (e.g., several) ketone moieties. In another morepreferred aspect, the ketone is acetone. See, for example, Qureshi andBlaschek, 2003, supra.

In another preferred aspect, the fermentation product is an organicacid. In another more preferred aspect, the organic acid is acetic acid.In another more preferred aspect, the organic acid is acetonic acid. Inanother more preferred aspect, the organic acid is adipic acid. Inanother more preferred aspect, the organic acid is ascorbic acid. Inanother more preferred aspect, the organic acid is citric acid. Inanother more preferred aspect, the organic acid is 2,5-diketo-D-gluconicacid. In another more preferred aspect, the organic acid is formic acid.In another more preferred aspect, the organic acid is fumaric acid. Inanother more preferred aspect, the organic acid is glucaric acid. Inanother more preferred aspect, the organic acid is gluconic acid. Inanother more preferred aspect, the organic acid is glucuronic acid. Inanother more preferred aspect, the organic acid is glutaric acid. Inanother preferred aspect, the organic acid is 3-hydroxypropionic acid.In another more preferred aspect, the organic acid is itaconic acid. Inanother more preferred aspect, the organic acid is lactic acid. Inanother more preferred aspect, the organic acid is malic acid. Inanother more preferred aspect, the organic acid is malonic acid. Inanother more preferred aspect, the organic acid is oxalic acid. Inanother more preferred aspect, the organic acid is propionic acid. Inanother more preferred aspect, the organic acid is succinic acid. Inanother more preferred aspect, the organic acid is xylonic acid. See,for example, Chen and Lee, 1997, Membrane-mediated extractivefermentation for lactic acid production from cellulosic biomass, Appl.Biochem. Biotechnol. 63-65: 435-448.

Recovery

The fermentation product(s) are optionally recovered after fermentationusing any method known in the art including, but not limited to,chromatography, electrophoretic procedures, differential solubility,distillation, or extraction. For example, alcohol, such as ethanol, isseparated from the fermented material and purified by conventionalmethods of distillation. Ethanol with a purity of up to about 96 vol. %can be obtained, which can be used as, for example, fuel ethanol,drinking ethanol, i.e., potable neutral spirits, or industrial ethanol.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Materials & Methods

Enzymes:

Trehalase (Ms37 tr or Ms trehalase) (P33WJF) from Myceliophthorasepedonium is shown as amino acids 21-697 in SEQ ID NO: 30 herein.

Trehalase (Cv37 tr or Cv trehalase) (P33W9X) from Chaetomium virescensis shown as amino acids 21-690 in SEQ ID NO: 4 herein.

Trehalase (Tr37 tr or Tr37 trehalase) (P337ZG) from Trichoderma reeseiis shown as SEQ ID NO: 12 in WO 2013/148993 and SEQ ID NO: 16 herein.

Trehalase (Tr65 tr or Tr65 trehalase) (P24TTB) from Trichoderma reeseiis shown as SEQ ID NO: 31 herein.

Cellulase VD: Cellulolytic composition derived from Trichoderma reeseicomprising GH61A polypeptide having cellulolytic enhancing activityderived from a strain of Penicillium emersonii (SEQ ID NO: 2 in WO2011/041397 or SEQ ID NO: 8 herein), Aspergillus fumigatusbeta-glucosidase (SEQ ID NO: 2 in WO 2005/047499 or SEQ ID NO: 6 herein)variant with the following substitutions: F100D, S283G, N456E, F512Y,disclosed in WO 2012/044915; Aspergillus fumigatus Cel7A CBH I disclosedas SEQ ID NO: 6 in WO2011/057140 and SEQ ID NO: 6 herein and Aspergillusfumigatus CBH II disclosed as SEQ ID NO: 18 in WO 2011/057140 and as SEQID NO: 8 herein.

Alpha-Amylase A (AAA): Bacillus stearothermophilus alpha-amylase withthe mutations I181*+G182*+N193F truncated to 491 amino acids (SEQ ID NO:18 herein).

Alpha-Amylase 369 (AA369): Bacillus stearothermophilus alpha-amylasewith the mutations:I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+Q254S+M284V truncated to491 amino acids (SEQ ID NO: 18).

Alpha-Amylase Blend B (AABB): Blend of Alpha-Amylase 369, GlucoamylasePoAMG498 and Pfu protease in a ratio of approximately 55:120:1 on μgEnzyme Protein basis.

Penicillium oxalicum glucoamylase variant PE498 (“PoAMG498”):Penicillium oxalicum glucoamylase variant having the followingmutations: K79V+P2N+P4S+P11F+T65A+Q327F (using SEQ ID NO: 23 herein fornumbering):

Protease Pfu (“PFU”): Protease derived from Pyrococcus furiosus shown inSEQ ID NO: 22 herein.

Glucoamylase E: comprises a blend comprising Talaromyces emersoniiglucoamylase disclosed in WO99/28448 (SEQ ID NO: 13 herein), Trametescingulata glucoamylase disclosed as SEQ ID NO: 2 in WO 06/69289 and SEQID NO: 14 herein, and Rhizomucor pusillus alpha-amylase with Aspergillusniger glucoamylase linker and SBD disclosed as SEQ ID NO: 15 herein withthe following substitutions: G128D+D143N (activity ratio AGU:AGU:FAU(F):approx. 30:7:1).

Glucoamylase U: Blend comprising Talaromyces emersonii glucoamylasedisclosed as SEQ ID NO: 34 in WO99/28448, Trametes cingulataglucoamylase disclosed as SEQ ID NO: 2 in WO 06/69289 and Rhizomucorpusillus alpha-amylase with Aspergillus niger glucoamylase linker andstarch binding domain (SBD) disclosed disclosed in SEQ ID NO: 15 hereinwith the following substitutions: G128D+D143N (activity ratio inAGU:AGU:FAU-F is about 65:15:1).

Glucoamylase A: Blend of Glucoamylase E and Cellulase VD in a ratio ofapproximately 10:3 on μg Enzyme Protein basis.

Yeast:

ETHANOL RED™ from Fermentis, USA

Sequence Identity

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the −nobrief option) is usedas the percent identity and is calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)Trehalase Assay:Principle:

Trehalose+H₂O

>2 Glucose

T=37° C., pH=5.7, A340 nm, Light path=1 cm

Spectrophotometric Stop Rate Determination

Unit Definition:

One unit will convert 1.0 mmole of trehalose to 2.0 mmoles of glucoseper minute at pH 5.7 at 37° C. (liberated glucose determined at pH 7.5).

(See Dahlqvist, A. (1968) Analytical Biochemistry 22, 99-107)

Protease Assays

AZCL-Casein Assay

A solution of 0.2% of the blue substrate AZCL-casein is suspended inBorax/NaH₂PO₄ buffer pH9 while stirring. The solution is distributedwhile stirring to microtiter plate (100 microL to each well), 30 microLenzyme sample is added and the plates are incubated in an EppendorfThermomixer for 30 minutes at 45° C. and 600 rpm. Denatured enzymesample (100° C. boiling for 20 min) is used as a blank. After incubationthe reaction is stopped by transferring the microtiter plate onto iceand the colored solution is separated from the solid by centrifugationat 3000 rpm for 5 minutes at 4° C. 60 microL of supernatant istransferred to a microtiter plate and the absorbance at 595 nm ismeasured using a BioRad Microplate Reader.

pNA-Assay

50 microL protease-containing sample is added to a microtiter plate andthe assay is started by adding 100 microL 1 mM pNA substrate (5 mgdissolved in 100 microL DMSO and further diluted to 10 mL withBorax/NaH₂PO₄ buffer pH 9.0). The increase in OD₄₀₅ at room temperatureis monitored as a measure of the protease activity.

Glucoamylase Activity (AGU)

Glucoamylase activity may be measured in Glucoamylase Units (AGU).

The Novo Glucoamylase Unit (AGU) is defined as the amount of enzyme,which hydrolyzes 1 micromole maltose per minute under the standardconditions 37° C., pH 4.3, substrate: maltose 23.2 mM, buffer: acetate0.1 M, reaction time 5 minutes.

An autoanalyzer system may be used. Mutarotase is added to the glucosedehydrogenase reagent so that any alpha-D-glucose present is turned intobeta-D-glucose. Glucose dehydrogenase reacts specifically withbeta-D-glucose in the reaction mentioned above, forming NADH which isdetermined using a photometer at 340 nm as a measure of the originalglucose concentration.

AMG incubation: Substrate: maltose 23.2 mM Buffer: acetate 0.1M pH: 4.30± 0.05 Incubation 37° C. ± 1 temperature: Reaction time: 5 minutesEnzyme working 0.5-4.0 AGU/mL range:

Color reaction: GlucDH:  430 U/L Mutarotase:   9 U/L NAD: 0.21 mMBuffer: phosphate 0.12M; 0.15M NaCl pH: 7.60 ± 0.05 Incubation 37° C. ±1 temperature: Reaction time:   5 minutes Wavelength:  340 nm

A folder (EB-SM-0131.02/01) describing this analytical method in moredetail is available on request from Novozymes A/S, Denmark, which folderis hereby included by reference.

Acid Alpha-Amylase Activity

When used according to the present invention the activity of an acidalpha-amylase may be measured in AFAU (Acid Fungal Alpha-amylase Units)or FAU-F.

Acid Alpha-Amylase Activity (AFAU)

Acid alpha-amylase activity may be measured in AFAU (Acid FungalAlpha-amylase Units), which are determined relative to an enzymestandard. 1 AFAU is defined as the amount of enzyme which degrades 5.260mg starch dry matter per hour under the below mentioned standardconditions.

Acid alpha-amylase, an endo-alpha-amylase(1,4-alpha-D-glucan-glucanohydrolase, E.C. 3.2.1.1) hydrolyzesalpha-1,4-glucosidic bonds in the inner regions of the starch moleculeto form dextrins and oligosaccharides with different chain lengths. Theintensity of color formed with iodine is directly proportional to theconcentration of starch. Amylase activity is determined using reversecolorimetry as a reduction in the concentration of starch under thespecified analytical conditions.

Standard conditions/reaction conditions: Substrate: Soluble starch,approx. 0.17 g/L Buffer: Citrate, approx. 0.03M Iodine (l2): 0.03 g/LCaCl₂: 1.85 mM pH: 2.50 ± 0.05 Incubation temperature: 40° C. Reactiontime: 23 seconds Wavelength: 590 nm Enzyme concentration: 0.025 AFAU/mLEnzyme working range: 0.01-0.04 AFAU/mL A folder EB-SM-0259.02/01describing this analytical method in more detail is available uponrequest to Novozymes A/S, Denmark, which folder is hereby included byreference.Determination of FAU-F

FAU-F Fungal Alpha-Amylase Units (Fungamyl) is measured relative to anenzyme standard of a declared strength.

Reaction conditions Temperature 37° C. pH 7.15 Wavelength 405 nmReaction time 5 min Measuring time 2 min

A folder (EB-SM-0216.02) describing this standard method in more detailis available on request from Novozymes A/S, Denmark, which folder ishereby included by reference.

Alpha-Amylase Activity (KNU)

The alpha-amylase activity may be determined using potato starch assubstrate. This method is based on the break-down of modified potatostarch by the enzyme, and the reaction is followed by mixing samples ofthe starch/enzyme solution with an iodine solution. Initially, ablackish-blue color is formed, but during the break-down of the starchthe blue color gets weaker and gradually turns into a reddish-brown,which is compared to a colored glass standard.

One Kilo Novo alpha amylase Unit (KNU) is defined as the amount ofenzyme which, under standard conditions (i.e., at 37° C.+/−0.05; 0.0003M Ca²⁺; and pH 5.6) dextrinizes 5260 mg starch dry substance MerckAmylum solubile.

A folder EB-SM-0009.02/01 describing this analytical method in moredetail is available upon request to Novozymes A/S, Denmark, which folderis hereby included by reference.

Alpha-Amylase Activity (KNU-A)

Alpha amylase activity is measured in KNU(A) Kilo Novozymes Units (A),relative to an enzyme standard of a declared strength.

Alpha amylase in samples and α-glucosidase in the reagent kit hydrolyzethe substrate (4,6-ethylidene(G₇)-p-nitrophenyl(G₁)-α,D-maltoheptaoside(ethylidene-G₇PNP) to glucose and the yellow-colored p-nitrophenol.

The rate of formation of p-nitrophenol can be observed by Konelab 30.This is an expression of the reaction rate and thereby the enzymeactivity.

The enzyme is an alpha-amylase with the enzyme classification number EC3.2.1.1.

Parameter Reaction conditions Temperature 37° C. pH 7.00 (at 37° C.)Substrate conc. Ethylidene-G₇PNP, R2: 1.86 mM Enzyme conc. 1.35-4.07KNU(A)/L (conc. of high/low standard in reaction mixture) Reaction time2 min Interval kinetic measuring time 7/18 sec. Wave length 405 nm Conc.of reagents/chemicals critical α-glucosidase, R1: ≥3.39 kU/L for theanalysis

A folder EB-SM-5091.02-D on determining KNU-A actitvity is availableupon request to Novozymes A/S, Denmark, which folder is hereby includedby reference.

pNP-G7 Assay

The alpha-amylase activity may be determined by a method employing theG7-pNP substrate. G7-pNP which is an abbreviation for4,6-ethylidene(G₇)-p-nitrophenyl(G₁)-α,D-maltoheptaoside, a blockedoligosaccharide which can be cleaved by an endo-amylase, such as analpha-amylase. Following the cleavage, the alpha-Glucosidase included inthe kit digest the hydrolysed substrate further to liberate a free PNPmolecule which has a yellow color and thus can be measured by visiblespectophometry at λ=405 nm (400-420 nm.). Kits containing G7-pNPsubstrate and alpha-Glucosidase is manufactured by Roche/Hitachi (cat.No. 11876473).

Reagents:

The G7-pNP substrate from this kit contains 22 mM 4,6-ethylidene-G7-pNPand 52.4 mM HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonicacid), pH 7.0).

The alpha-Glucosidase reagent contains 52.4 mM HEPES, 87 mM NaCl, 12.6mM MgCl₂, 0.075 mM CaCl₂, ≥4 kU/L alpha-glucosidase).

The substrate working solution is made by mixing 1 mL of thealpha-Glucosidase reagent with 0.2 mL of the G7-pNP substrate. Thissubstrate working solution is made immediately before use.

Dilution buffer: 50 mM MOPS, 0.05% (w/v) Triton X100 (polyethyleneglycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether (C₁₄H₂₂O(C₂H₄O),(n=9-10))), 1 mM CaCl2, pH8.0.

Procedure:

The amylase sample to be analyzed is diluted in dilution buffer toensure the pH in the diluted sample is 7. The assay is performed bytransferring 20 μl diluted enzyme samples to 96 well microtiter plateand adding 80 μl substrate working solution. The solution is mixed andpre-incubated 1 minute at room temperature and absorption is measuredevery 20 sec. over 5 minutes at OD 405 nm.

The slope (absorbance per minute) of the time dependent absorption-curveis directly proportional to the specific activity (activity per mgenzyme) of the alpha-amylase in question under the given set ofconditions. The amylase sample should be diluted to a level where theslope is below 0.4 absorbance units per minute.

Phadebas Activity Assay

The alpha-amylase activity may also be determined by a method using thePhadebas substrate (from for example Magle Life Sciences, Lund, Sweden).A Phadebas tablet includes interlinked starch polymers that are in theform of globular microspheres that are insoluble in water. A blue dye iscovantly bound to these microspheres. The interlinked starch polymers inthe microsphere are degraded at a speed that is proportional to thealpha-amylase activity. When the alpha-amylse degrades the starchpolymers, the released blue dye is water soluble and concentration ofdye can be determined by measuring absorbance at 620 nm. Theconcentration of blue is proportional to the alpha-amylase activity inthe sample.

The amylase sample to be analysed is diluted in activity buffer with thedesired pH. One substrate tablet is suspended in 5 mL activity bufferand mixed on magnetic stirrer. During mixing of substrate transfer 150μl to microtiter plate (MTP) or PCR-MTP. Add 30 μl diluted amylasesample to 150 μl substrate and mix. Incubate for 15 minutes at 37° C.The reaction is stopped by adding 30 μl M NaOH and mix. Centrifuge MTPfor 5 minutes at 4000×g. Transfer 100 μl to new MTP and measureabsorbance at 620 nm.

The amylase sample should be diluted so that the absorbance at 620 nm isbetween 0 and 2.2, and is within the linear range of the activity assay.

Reducing Sugar Activity Assay:

The alpha-amylase activity may also be determined by reducing sugarassay with for example corn starch substrate. The number of reducingends formed by the alpha-amylase hydrolysing the alpha-1,4-glycosidiclinkages in starch is determined by reaction with p-Hydroxybenzoic acidhydrazide (PHBAH). After reaction with PHBAH the number of reducing endscan be measured by absorbance at 405 nm and the concentration ofreducing ends is proportional to the alpha-amylase activity in thesample.

The corns starch substrate (3 mg/ml) is solubilised by cooking for 5minutes in milliQ water and cooled down before assay. For the stopsolution prepare a Ka-Na-tartrate/NaOH solution (K—Na-tartrate (Merck8087) 50 g/l, NaOH 20 g/l) and prepare freshly the stop solution byadding p-Hydroxybenzoic acid hydrazide (PHBAH, Sigma H9882) toKa-Na-tartrate/NaOH solution to 15 mg/ml.

In PCR-MTP 50 μl activity buffer is mixed with 50 μl substrate. Add 50μl diluted enzyme and mix. Incubate at the desired temperature in PCRmachine for 5 minutes. Reaction is stopped by adding 75 μl stop solution(Ka-Na-tartrate/NaOH/PHBAH). Incubate in PCR machine for 10 minutes at95° C. Transfer 150 μl to new MTP and measure absorbance at 405 nm.

The amylase sample should be diluted so that the absorbance at 405 nm isbetween 0 and 2.2, and is within the linear range of the activity assay.

EnzChek® Assay:

For the determination of residual amylase activity an EnzChek® UltraAmylase Assay Kit (E33651, Invitrogen, La Jolla, Calif., USA) may beused.

The substrate is a corn starch derivative, DQ™ starch, which is cornstarch labeled with BODIPY® FL dye to such a degree that fluorescence isquenched. One vial containing approx. 1 mg lyophilized substrate isdissolved in 100 microliters of 50 mM sodium acetate (pH 4.0). The vialis vortexed for 20 seconds and left at room temperature, in the dark,with occasional mixing until dissolved. Then 900 microliters of 100 mMacetate, 0.01% (w/v) TRITON® X100, 0.125 mM CaCl₂, pH 5.5 is added,vortexed thoroughly and stored at room temperature, in the dark untilready to use. The stock substrate working solution is prepared bydiluting 10-fold in residual activity buffer (100 mM acetate, 0.01%(w/v) TRITON® X100, 0.125 mM CaCl₂, pH 5.5). Immediately afterincubation the enzyme is diluted to a concentration of 10-20 ng enzymeprotein/ml in 100 mM acetate, 0.01% (W/v) TRITON® X100, 0.125 mM CaCl₂,pH 5.5.

For the assay, 25 microliters of the substrate working solution is mixedfor 10 second with 25 microliters of the diluted enzyme in a black 384well microtiter plate. The fluorescence intensity is measured(excitation: 485 nm, emission: 555 nm) once every minute for 15 minutesin each well at 25° C. and the V_(max) is calculated as the slope of theplot of fluorescence intensity against time. The plot should be linearand the residual activity assay has been adjusted so that the dilutedreference enzyme solution is within the linear range of the activityassay.

Determination of Pullulanase Activity (NPUN)

Endo-pullulanase activity in NPUN is measured relative to a Novozymespullulanase standard. One pullulanase unit (NPUN) is defined as theamount of enzyme that releases 1 micro mol glucose per minute under thestandard conditions (0.7% red pullulan (Megazyme), pH 5, 40° C., 20minutes). The activity is measured in NPUN/ml using red pullulan.

1 mL diluted sample or standard is incubated at 40° C. for 2 minutes.0.5 mL 2% red pullulan, 0.5 M KCl, 50 mM citric acid, pH 5 are added andmixed. The tubes are incubated at 40° C. for 20 minutes and stopped byadding 2.5 ml 80% ethanol. The tubes are left standing at roomtemperature for 10-60 minutes followed by centrifugation 10 minutes at4000 rpm. OD of the supernatants is then measured at 510 nm and theactivity calculated using a standard curve.

Media and Solutions

YP+2% glucose medium was composed of 1% yeast extract, 2% peptone and 2%glucose.

YP+2% maltodextrin medium was composed of 1% yeast extract, 2% peptoneand 2% maltodextrin.

PDA agar plates were composed of potato infusion (potato infusion wasmade by boiling 300 g of sliced (washed but unpeeled) potatoes in waterfor 30 minutes and then decanting or straining the broth throughcheesecloth. Distilled water was then added until the total volume ofthe suspension was one liter, followed by 20 g of dextrose and 20 g ofagar powder. The medium was sterilized by autoclaving at 15 psi for 15minutes (Bacteriological Analytical Manual, 8th Edition, Revision A,1998).

LB plates were composed of 10 g of Bacto-Tryptone, 5 g of yeast extract,10 g of sodium chloride, 15 g of Bacto-agar, and deionized water to 1liter.

LB medium was composed of 10 g of Bacto-Tryptone, 5 g of yeast extract,10 g of sodium chloride, and deionized water to 1 liter.

COVE sucrose plates were composed of 342 g of sucrose, 20 g of agarpowder, 20 ml of COVE salts solution, and deionized water to 1 liter.The medium was sterilized by autoclaving at 15 psi for 15 minutes(Bacteriological Analytical Manual, 8th Edition, Revision A, 1998). Themedium was cooled to 60° C. and 10 mM acetamide, 15 mM CsCl, TRITON®X-100 (50 μl/500 ml) were added.

COVE salts solution was composed of 26 g of MgSO4.7H2O, 26 g of KCL, 26g of KH2PO4, 50 ml of COVE trace metals solution, and deionized water to1 liter.

COVE trace metals solution was composed of 0.04 g of Na2B4O7.10H2O, 0.4g of CuSO4.5H2O, 1.2 g of FeSO4.7H2O, 0.7 g of MnSO4.H2O, 0.8 g ofNa2MoO4.2H2O, 10 g of ZnSO4.7H2O, and deionized water to 1 liter.

Example 1

Cloning of Chaetomium virescens Trehalase (Cv37 tr) Polypeptide (P33W9X)Coding Sequence from Chaetomium virescens CBS547.75.

The Chaetomium virescens trehalase (Cv Trehalase) polypeptide codingsequence was cloned from Chaetomium virescens CBS547.75 DNA by PCR.

Chaetomium virescens CBS547.75 was purchased from the Centraalbureauvoor Schimmelcultures (Utrecht, the Netherlands). The fungal strain wascultivated in 100 ml of YP+2% glucose medium in 1000 ml Erlenmeyer shakeflasks for 5 days at 20° C. Mycelia were harvested from the flasks byfiltration of the medium through a Buchner vacuum funnel lined withMIRACLOTH® (EMD Millipore, Billerica, Mass., USA). Mycelia were frozenin liquid nitrogen and stored at −80° C. until further use. Genomic DNAwas isolated using a DNEASY® Plant Maxi Kit (QIAGEN GMBH, HildenGermany) according to the manufacturer's instructions.

Genomic sequence information was generated by Illumina MySeq (IlluminaInc., San Diego, Calif.). 5 μgs of the isolated Chaetomium virescensgenomic DNA was used for library preparation and analysis according tothe manufacturer's instructions. Genes were called using GeneMark.hmm ESversion 2.3c and identification of the catalytic domain was made using“Trehalase PF01204” Hidden Markov Model provided by Pfam. Thepolypeptide coding sequence for the entire coding region was cloned fromChaetomium virescens CBS547.75 genomic DNA by PCR using the primers (SEQID NO: 24 and SEQ ID NO: 25) described below.

KKSC0334-F (SEQ ID NO: 24)5′- ACACAACTGGGGATCCACCATGACGCTCCGACACCTCGG -3′ KKSC0334-R(SEQ ID NO: 25) 5′- CTAGATCTCGAGAAGCTT TCACGACCTCCTCCCTACCC -3′

Bold letters represent Chaetomium virescens enzyme coding sequence.Restriction sites are underlined. The sequence to the left of therestriction sites is homologous to the insertion sites of pDau109 (WO2005/042735).

The amplification reaction (50 μls) was performed according to themanufacturer's instructions (Phusion HiFi DNA polymerase cat # M0530S,New England Biolabs Inc.) with the following final concentrations:

PCR mix 5x HF Phusion Buffer 10 ul dNTP 10 mM 1 ul DMSO 1.5 ul Primer F1 ul Prime R 1 ul Phusion polymerase 0.5 uls H2O 35 uls Genomic DNA 1uls Total volume 50 uls

The PCR reaction was incubated in a DYAD® Dual-Block Thermal Cycler(BioRad, USA) programmed for 1 cycle at 98° C. for 30 seconds; 35 cycleseach at 98° C. for 10 seconds, 72° C. for 2 minutes followed by 1 cycleat 72° C. for 7 minutes. Samples were cooled to 10° C. before removaland further processing.

Four μl of the PCR reaction was analyzed by 1% agarose gelelectrophoresis using 40 mM Tris base, 20 mM sodium acetate, 1 mMdisodium EDTA (TAE) buffer. A major band of about 2241 bp was observed.The remaining PCR reaction was purified directly with an ILLUSTRA™ GFX™PCR DNA and Gel Band Purification Kit (GE Healthcare, Piscataway, N.J.,USA) according to the manufacturer's instructions.

Two μg of plasmid pDau109 was digested with Bam HI and Hind III and thedigested plasmid was run on a 1% agarose gel using 50 mM Tris base-50 mMboric acid-1 mM disodium EDTA (TBE) buffer in order to remove thestuffer fragment from the restricted plasmid. The bands were visualizedby the addition of SYBR® Safe DNA gel stain (Life TechnologiesCorporation, Grand Island, N.Y., USA) and use of a 470 nm wavelengthtransilluminator. The band corresponding to the restricted plasmid wasexcised and purified using an ILLUSTRA™ GFX™ PCR DNA and Gel BandPurification Kit (Cat. no 28-9034-70 from GE Healthcare). The plasmidwas eluted into 10 mM Tris pH 8.0 and its concentration adjusted to 20ng per μl. An IN-FUSION® HD EcoDry-Down PCR Cloning Kit (BD Biosciences,Palo Alto, Calif., USA), was used to clone the 2241 bp PCR fragment intopDau109 digested with Bam HI and Hind III (20 ng). The IN-FUSION® totalreaction volume was 10 μl. The IN-FUSION® total reaction volume was 10μl. The IN-FUSION® reaction was transformed into FUSION-BLUE™ E. colicells (BD Biosciences, Palo Alto, Calif., USA) according to themanufacturer's protocol and plated onto LB agar plates supplemented with50 μg of ampicillin per ml. After incubation overnight at 37° C.,transformant colonies were observed growing under selection on the LBplates supplemented with 50 μg of ampicillin per ml.

One plasmid with the correct C. virescens trehalase coding sequence (SEQID NO: 3) was chosen. The plasmid was designated pKKSC0334-1. Cloning ofthe Chaetomium virescens trehalase gene into Bam HI-HindIII digestedpDau109 resulted in transcription of the C. virescens trehalase geneunder the control of a NA2-tpi double promoter. NA2-tpi is a modifiedpromoter from an Aspergillus niger neutral alpha-amylase gene in whichthe untranslated leader has been replaced by an untranslated leader froman Aspergillus nidulans triose phosphate isomerase gene.

The expression plasmid pKKSC0334-1 was transformed into protoplasts ofAspergillus oryzae MT3568 according to the method of European Patent EP0238023, pages 14-15. Aspergillus oryzae MT3568 is an amdS (acetamidase)disrupted derivative of A. oryzae JaL355 (WO 2002/40694) in which pyrGauxotrophy was restored in the process of inactivating the A. oryzaeamdS gene.

E. coli 3701 containing pKKSC0334-1 was grown overnight according to themanufacturer's instructions (Genomed) and plasmid DNA of pKKSC0334-1 wasisolated using a Plasmid Midi Kit (Genomed JETquick kit, cat. nr.400250, GENOMED GmbH, Germany) according to the manufacturer'sinstructions. The purified plasmid DNA was transformed into Aspergillusoryzae MT3568. A. oryzae MT3568 protoplasts were prepared according tothe method of Christensen et al., 1988, Bio/Technology 6: 1419-1422. Theselection plates consisted of COVE sucrose with +10 mM acetamide +15 mMCsCl+TRITON® X-100 (50 μl/500 ml). The plates were incubated at 37° C.Briefly, 8 uls of plasmid DNA representing 3 ugs of DNA was added to 100uls MT3568 protoplasts. 250 ul of 60% PEG solution was added and thetubes were gently mixed and incubate at 37° C. for 30 minutes. The mixwas added to 10 ml of pre melted Cove top agarose (The top agarosemelted and then the temperature equilibrated to 40° C. in a warm waterbath before being added to the protoplast mixture). The combined mixturewas then plated on two Cove-sucrose selection petri plates with 10 mMAcetamide. The plates are incubated at 37° C. for 4 days. SingleAspergillus transformed colonies were identified by growth on theselection Acetimide as a carbon source. Each of the four A. oryzaetransformants were inoculated into 750 μl of YP medium supplemented with2% glucose and also 750 μl of 2% maltodextrin and also DAP4C in 96 welldeep plates and incubated at 37° C. stationary for 4 days. At same timethe four transformants were restreaked on COVE-2 sucrose agar medium.

Culture broth from the Aspergillus oryzae transformants were thenanalyzed for production of the P33W9X trehalase polypeptide by SDS-PAGEusing NUPAGE® 10% Bis-Tris SDS gels (Invitrogen, Carlsbad, Calif., USA)according to the manufacturer. A band at approximately 75 kDa wasobserved for each of the Aspergillus oryzae transformants. One A. oryzaetransformant producing the P33W9X polypeptide was designated A. oryzaeEXP09256.

For larger scale production, A. oryzae EXP09256 spores were spread ontoa PDA plate and incubated for five days at 37° C. The confluent sporeplate was washed twice with 5 ml of 0.01% TWEEN® 20 to maximize thenumber of spores collected. The spore suspension was then used toinoculate nine 500 ml flasks containing 100 ml of Dap-4C medium. Thecultures were incubated at 30° C. with constant shaking at 150 rpm. Atday four post-inoculation, the culture broth was collected by filtrationthrough a bottle top MF75™ SUPOR® MachV 0.2 μm PES filter (Thermo FisherScientific, Roskilde, Denmark). Fresh culture broth from thistransformant produced a band of GH37 protein of approximately 75 kDa(designated EXP09256). The identity of this band as the C. virescenstrehalase polypeptide was verified by peptide sequencing.

Characterization of the EXP09256 trehalase polypeptide coding sequencefrom Chaetomium virescens CBS547.75

The genomic DNA sequence and deduced amino acid sequence of theChaetomium virescens CBS547.75.

The Chaetomium virescens trehalase polypeptide (P33W9X) genomic codingsequence is shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. Theencoded predicted protein is 690 amino acids. Using the SignalP program(Nielsen et al., 1997, supra), a signal peptide of 20 residues waspredicted. The predicted mature protein contains 670 amino acids with apredicted molecular mass of 75 kDa and a predicted isoelectric point of5.5.

Example 2

Expression of Trehalase (P33WJF) from Myceliophthora sepedonium (MsTr37)

This strain was purchased from UAMH and received on the 12 Sep. 1988 asUAMH5004. UAMH is the University of Alberta Microfungus Collection andHerbarium.

Myceliophthora sepedonium strain UAHM5004 was obtained from theUniversity of Alberta Microfungus Collection and Herbarium UAMH,Edmonton, Alberta, Canada T6G 2R3. The earlier genus and speciesdesignation Cornyascus sepedonium is still employed.

The trehalase polypeptide (P33WJF) coding sequence was cloned fromMyceliophthora sepedonium strain UAHM5004 DNA by PCR.

The fungal strain was cultivated, DNA isolated genome sequenced and thetrehalase enzyme candidate identified as in example 1.

The polypeptide coding sequence for the entire coding region was clonedfrom Myceliophthora sepedonium strain UAHM5004 genomic DNA by PCR usingthe primers (SEQ ID NO: 26 and SEQ ID NO: 27) described below.

KKSC0335-F (SEQ ID NO: 26)5′- ACACAACTGGGGATCCACCATGGCGCTACGACACATCGC-3′ KKSC0335-R(SEQ ID NO: 27) 5′- CTAGATCTCGAGAAGCTT TTACGAGACGGAGACACTAAACA

Bold letters represent Myceliophthora sepedonium enzyme coding sequence.Restriction sites are underlined. The sequence to the left of therestriction sites is homologous to the insertion sites of pDau109 (WO2005/042735).

The amplification reaction (50 μls) was performed according to theprotocol in example 1 according the manufacturer's instructions (PhusionHiFi DNA polymerase cat # M0530S, New England Biolabs Inc

Four μl of the PCR reaction was analyzed by 1% agarose gelelectrophoresis using 40 mM Tris base, 20 mM sodium acetate, 1 mMdisodium EDTA (TAE) buffer. A major band of about 2354 bp was observed.The remaining PCR reaction was purified directly with an ILLUSTRA™ GFX™PCR DNA and Gel Band Purification Kit (GE Healthcare, Piscataway, N.J.,USA) and cloned into pDau109 as in example 1.

One plasmid with the correct M. sepedonium trehalase coding sequence(SEQ ID NO: x) was chosen. The plasmid was designated pKKSC0335-1. Theexpression plasmid pKKSC0335-1 was transformed into protoplasts ofAspergillus oryzae MT3568 and a transformants chosen and propagated alsoaccording example 1.

Production of the trehalase polypeptide (P33WJF) by SDS-PAGE usingNUPAGE® 10% Bis-Tris SDS gels (Invitrogen, Carlsbad, Calif., USA)according to the manufacturer. A band at approximately 76 kDa wasobserved for each of the Aspergillus oryzae transformants. One A. oryzaetransformant producing the P33W9X polypeptide was designated A. oryzaeEXP09258.

Larger scale production, A. oryzae EXP09258 was performed also accordingto Example 1.

Characterization of the EXP09258 trehalase polypeptide coding sequencefrom Myceliophthora sepedonium strain UAHM5004 The genomic DNA sequenceand deduced amino acid sequence of the Myceliophthora sepedonium strainUAHM5004

Trehalase polypeptide (P33WJF) genomic coding sequence are shown in SEQID NO: 1 and SEQ ID NO: 2, respectively. The encoded predicted proteinis 697 amino acids. Using the SignalP program (Nielsen et al., 1997,supra), a signal peptide of 20 residues was predicted. The predictedmature protein contains 677 amino acids with a predicted molecular massof 76 kDa and a predicted isoelectric point of 5.1.

Example 3

Glucose Production with Trehalase Addition Using Supernatant of FermDrop

Ferm drop sample at the end of fermentation from industrial corn ethanolplant was first centrifuged, and supernatant was collected for thetesting. The trehalose level in the supernatant was analyzed to bearound 2 g/L by an Ion Chromatography assay. The pH of the supernatantwas measured as 4.6, and LACTROL® was supplemented as antibacterialagent at the level of 3 ppm. Next, 2 ml of supernatant was aliquotedinto 15 ml polypropylene tube. Each tube was dosed with trehalase listedin Table 1. The enzyme dosage was 0, 0.05, 0.5 and 5 ug/ml,respectively. Each treatment ran three replicate. Afterwards, all tubeswere incubated in 32° C. water bath for 5 hours. Samples were taken at 5hour of incubation for HPLC analysis. The HPLC preparation consisted ofstopping the reaction by addition of 20 micro liters of 40% H₂SO₄, andfiltering through a 0.45 micrometer filter. Agilent™ 1100 HPLC systemcoupled with RI detector was used to determine glucose concentration.The separation column was aminex HPX-87H ion exclusion column (300mm×7.8 mm) from BioRad™.

TABLE 1 List of trehalase tested Purified ID Sequence and ID Code Donorsource U3EAN SEQ ID NO: 30 - P33WJG Myceliophthora sepidonium (Ms37 tr)U3EAJ SEQ ID NO: 4 - P33W9X Chaetomium virescens (Cv37 tr) U3EAH SEQ IDNO: 16 - P337ZG Trichoderma reesei (Tr37 tr)Results

The glucose results from HPLC analysis is summarized in FIG. 1. Comparedto control without any trehalase addition, all treatments with trehalaseshowed glucose increase. The amount of glucose increase was depended onthe dosage of trehalase and its source. Results showed Myceliophthorasepidonium trehalase (Ms trehalase) performed the best. At dosage of 0.5ug/ml, all the trehalose was converted into glucose with 5 hourincubation. Chaetomium virescens trehalase was a little less efficientthan Myceliophthora sepidonium trehalase, but still much better thanTrichoderma reesei trehalase (Tr tr).

Example 4

Application of Ms Trehalase in Conventional SSF Process for EthanolProduction

All treatments were evaluated via 5 g small assay. Each treatment ranfive replicate. Three corn mashes liquefied by Alpha-Amylase Blend B(AABB) (Mash A and B) and Alpha-Amylase A (AAA) (Mash C) from industrialcorn ethanol plants were used for the testing. 3 ppm penicillin and 1000ppm urea were supplemented into each mash. The pH of the slurries wasadjusted to 5.0 with 40% H₂SO₄ or 50% NaOH. Approximately 5 g of theslurry was added to 15 ml polypropylene tube. The tubes were prepared bydrilling a 1/32 inch hole and the empty tubes were then weighed beforecorn slurry was added. The tubes were weighed again after mash was addedto determine the exact weight of mash in each tube. Each tube was dosedwith actual enzyme dosage based on the exact weight of corn slurry ineach tube. The enzyme dosage for each treatment was listed in Table 2.Trehalase from Myceliophthora sepidonium (Ms37 trehalase) (SEQ ID NO:30) was used in this study. Afterwards, the tubes were dosed with 50 ulof yeast propagate to around 5 g corn mash, and then were incubated in32° C. water batch for SSF. Samples were taken at 53 hour offermentation for HPLC analysis. The HPLC preparation consisted ofstopping the reaction by addition of 50 micro liters of 40% H₂50₄,centrifuging, and filtering through a 0.45 micrometer filter. Agilent™1100 HPLC system coupled with RI detector was used to determine sugars,acids and ethanol concentration. The separation column was aminexHPX-87H ion exclusion column (300 mm×7.8 mm) from BioRad™.

TABLE 2 AMG and trehalase dosage for each treatment in SSF AMG,Trehalase, Mash AMG AGU/g-DS ug EP/g-DS 1 Mash A Glucoamylase U 0.56 0 25 3 Glucoamylase E 0.60 0 4 5 5 Glucoamylase A 0.60 0 6 5 7 Mash BGlucoamylase U 0.56 0 8 5 9 Glucoamylase E 0.60 0 10 5 11 Glucoamylase A0.60 0 12 5 13 Mash C GlucoamylaseU 0.56 0 14 5 15 Glucoamylase E 0.60 016 5 17 Glucoamylase A 0.60 0 18 5Results

The results from HPLC analysis were summarized in Table 3. Compared tothe control, treatment with trehalase addition achieved much higherethanol titer, ranging from between 0.5 to 1.2% yield increase.

TABLE 3 Ethanol yield increase with trehalase addition compared to thecontrol in SSF Treatment Mash A Mash B Mash C Glucoamylase U (control) —— — Glucoamylase U + 0.70% 0.64% 0.90% Ms37 trehalase Glucoamylase E(control) — — — Glucoamylase E + 1.13% 0.91% 1.16% Ms37 trehalaseGlucoamylase A (control) — — — Glucoamylase A + 0.54% 0.64% 1.01% Ms37trehalase

Example 5

Purification of GH37 Trehalases

Filtered culture broth from fermentation of A. oryzae harboring the GH37trehalase gene was added solid ammonium sulfate to a final concentrationof 2 M and pH adjusted to 7. The solution, containing the trehalase, wasapplied to a hydrophobic interaction column (butyl Toyopearl,approximately 50 ml in a XK26 column, equilibrated with buffer A), usingas buffer A 50 mM Hepes+2 M ammonium sulfate pH 7.0, and as buffer B 50mM Hepes pH 7.0. Unbound material was washed off the column withequilibration buffer and the trehalase was eluted with a linear gradient(100% to 0% A) over 5 column volumes and 10 ml fractions were collected.Based on the chromatogram trehalase containing fractions were pooled anddialyzed against a large volume of 20 mM Hepes pH 7.0.

The purified GH37 trehalases are listed in the table below:

Purified Protein Sequence Sample ID Donor Scientific Name Reference IDU3EAN Myceliophthora sepedonium SEQ ID NO: 30 - P33WJF U3EAJ Chaetomiumvirescens SEQ ID NO: 4 - P33W9X U3EAH Trichoderma reesei SEQ ID NO: 16 -P337ZGMolecular Weight of the Purified Trehalases

The molecular weight, as estimated from SDS-PAGE, was approximately 100kDa for all three trehalases and the purity was in all cases >95%.

Example 6

Purification of a GH65 Trehalase from Trichoderma reesei (SEQ ID NO: 31)

Filtered culture broth from fermentation of A. oryzae harboring the GH65trehalase gene from Trichoderma reesei (P24TTB) was loaded on a gelfiltration column (about 780 ml Sephadex G-25 medium) equilibrated with50 mm Na-acetate pH 5.0 and fractions collected. Based on thechromatogram fractions containing protein (A280>A260) that ran straightthrow the column were pooled. The trehalase containing pool wasconcentrated using a Viva cell with a 30 kDa cut-off membrane.

Molecular Weight of the Purified Trehalases

The molecular weight, as estimated from SDS-PAGE, was approximately 120kDa and the purity was >95%.

Example 7

Determination of Trehalase Activity (GOD)

GOD-Perid

50 microliter trehalase-containing enzyme solution (appropriatelydiluted with 20 mM MES buffer pH 5.0) is dispensed in a microtiter platewell (e.g. NUNC 269620) and 50 microliter substrate (10 mg/ml trehalosein 20 mM MES buffer pH 5.0) is added. The plate is sealed and incubated15 min., shaken with 750 rpm at 32° C. After the incubation 20microliter of reaction solution is transferred to an empty microtiterplate and 180 microliter GOD-perid [600 ppm glucose oxidase (SigmaG1625), 20 ppm peroxidase (Sigma P8125), 0.1% ATBS (Roche 102946) in 0.1M K-phosphate buffer pH 7.0] is added. The plate is left for 30 minutesat 20-30° C. and following this the absorbance at 420 nm is measured ina microtiter plate spectrophotometer.

Example 8

pH Profile

The pH profile was determined at 32° C. in the pH range of 2.0 to 7.5(in 0.5 pH-unit steps) as described above in the section “Determinationof trehalase activity, GOD-perid”, except that a buffer cocktail (110 mMacetic acid, 110 mM citric acid and 110 mM MES) was used instead of the20 mM MES buffer pH 5.0 buffer. The results are summarized in table 5below. The values given for each pH in the range of 2.0-7.5 are therelative activity in % normalized to the value at optimum.

Relative activity pH 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 Tr37 tr SEQ ID36 100 98 68 35 18 16 14 15 13 10 7 NO: 16 U3EAH Cv37 tr SEQ ID 27 73 81100 86 77 70 76 90 77 53 25 NO: 4 U3EAJ Ms37 tr SEQ ID 26 85 88 100 5952 52 46 2 32 24 18 NO: 30 U3EAN

Example 9

Thermostability Using DSC Data

The thermostability of trehalases were determined by DifferentialScanning calorimetry (DSC) using a VP-capillary DSC instrument (MicroCalInc., Piscataway, N.J., USA) equipped with an auto sampler. Aliquots ofthe trehalase, purified as described in Example 1, were buffer-changed(see buffer in table below) using prepacked NAP-5 columns. The sampleswere diluted with the corresponding buffer to approximately 0.5 mg/mland the buffer was used as reference solution. Sample and referencesolutions (approx. 0.5 ml) were thermally pre-equilibrated for 10minutes at 20° C. and the DSC scan was performed from 20 to 100° C. at ascan rate of 200 K/hour. Data-handling was performed using the MicroCalOrigin software (version 7.0383). The thermal denaturation temperature,Td (° C.), was taken as the top of denaturation peak (major endothermicpeak) in thermograms (Cp vs. T) obtained after heating the trehalasesolution in the buffer at a constant programmed heating rate.Denaturation temperatures were determined at an accuracy ofapproximately +/−0.5° C.

The results of the DSC measurements are summarized in the table below.

Purified Protein Sequence Sample Donor SEQ ID and ID Scientific NameReference ID Buffer Td (° C.) U3EAN Myceliophthora SEQ ID NO: 30 50 mMMES 68.9 sepedonium P33WJF pH 5.0 U3EAJ Chaetomium SEQ ID NO: 4 50 mMMES 65.1 virescens P33W9X pH 5.0 U3EAH Trichoderma SEQ ID NO: 16 50 mMMES 67.0 reesei P337ZG pH 5.0 U6B9P Trichoderma SEQ ID NO: 31 50 mM MES64.7 reesei P24TTB pH 5.0

Example 10

Glucose Production by Ms37 Trehalase (SEQ ID NO: 30) and Tr65 Trehalase(SEQ ID NO: 31)

Ferm drop samples at the end of fermentation from two industrial cornethanol plants were collected and pH was measured as 4.68 (A) and 4.49(B) respectively. Ferm drop samples were centrifuged at 3500 rpm for 15minutes, and then supernatants were collected and used for the trehalaseapplication test. First, penicillium was supplemented as antibacterialagent at the level of 3 ppm. Next, 2 ml of supernatant was aliquotedinto 15 ml polypropylene tube. Each tube was dosed with trehalase listedin the table below. The enzyme doses tested were 0, 0.05, 0.15, 0.5 and5 ug per ml of supernatant. Each treatment ran three replicate.Afterwards, all tubes were incubated in 32° C. water bath for 5 hours.Samples were taken at 5 hour of incubation for HPLC analysis. The HPLCpreparation consisted of stopping the reaction by addition of 20 microliters of 40% H2SO4, and filtering through a 0.45 micrometer filter.Agilent™ 1100 HPLC system coupled with RI detector was used to determineglucose concentration. The separation column was aminex HPX-87H ionexclusion column (300 mm×7.8 mm) from BioRad™.

Trehalases Tested

Purified ID GH family Donor source and SEQ ID U3EAN GH37 Myceliophthorasepidonium (SEQ ID NO: 30) U6BP9 GH65 Trichoderma reesei (SEQ ID NO: 31)Results

The glucose results from HPLC analysis were summarized by JMP in FIG. 2.Compared to control without any trehalase addition, treatments withtrehalase showed glucose increase, except the lowest dose of 0.05 ug/mlin Ferm Drop B. The amount of glucose increase was dependent on thedosage of trehalase and its source. Results showed that Myceliophthorasepidonium GH37 trehalase (SEQ ID NO: 30) performed better thanTrichoderma reesei GH65 trehalase (SEQ ID NO: 31). At high dose of 5ug/ml trehalase addition, the trehalose in ferm drop samples wascompletely converted to glucose by the trehalases.

The present invention is presented in the following numbered paragraphs:

1. A polypeptide having trehalase activity, selected from the groupconsisting of:

-   -   (a) a polypeptide having at least 90% sequence identity to the        mature polypeptide of SEQ ID NO: 30 or SEQ ID NO: 2 or at least        80% sequence identity to the mature polypeptide of SEQ ID NO: 4;    -   (b) a polypeptide encoded by a polynucleotide that hybridizes        under high stringency conditions with (i) the mature polypeptide        coding sequence of SEQ ID NO: 29, SEQ ID NO: 1 or SEQ ID NO:        3, (ii) the cDNA sequence thereof, or (iii) the full-length        complement of (i) or (ii);    -   (c) a polypeptide encoded by a polynucleotide having at least        60% sequence identity to the mature polypeptide coding sequence        of SEQ ID NO: 29 or SEQ ID NO: 1 or SEQ ID NO: 3 or the cDNA        sequence thereof;    -   (d) a variant of the mature polypeptide of SEQ ID NO: 30, SEQ ID        NO: 2 or SEQ ID NO: 4 comprising a substitution, deletion,        and/or insertion at one or more (e.g., several) positions; and    -   (e) a fragment of the polypeptide of (a), (b), (c), or (d) that        has trehalase activity.        2. The polypeptide of paragraph 1, having at least 91%, at least        92%, at least 93%, at least 94%, at least 95%, at least 96%, at        least 97%, at least 98%, at least 99% or 100% sequence identity        to the mature polypeptide of SEQ ID NO: 30 or SEQ ID NO: 2.        3. The polypeptide of paragraph 1 or 2, which is encoded by a        polynucleotide that hybridizes under very high stringency        conditions with (i) the mature polypeptide coding sequence of        SEQ ID NO: 29 or SEQ ID NO: 1, (ii) the cDNA sequence thereof,        or (iii) the full-length complement of (i) or (ii).        4. The polypeptide of any of paragraphs 1-3, which is encoded by        a polynucleotide having at least 70%, at least 75%, at least        80%, at least 85%, at least 90%, at least 91%, at least 92%, at        least 93%, at least 94%, at least 95%, at least 96%, at least        97%, at least 98%, at least 99% or 100% sequence identity to the        mature polypeptide coding sequence of SEQ ID NO: 29 or SEQ ID        NO: 1 or the cDNA sequence thereof.        5. The polypeptide of any of paragraphs 1-4, comprising or        consisting of SEQ ID NO: 30 or SEQ ID NO: 2 or the mature        polypeptide of SEQ ID NO: 30 shown as amino acids 21-694 or SEQ        ID NO: 2 shown as amino acids 21-697 of SEQ ID NO: 2.        6. The polypeptide of any of paragraphs 1-6, which is a variant        of the mature polypeptide of SEQ ID NO: 30 or SEQ ID NO: 2        comprising a substitution, deletion, and/or insertion at one or        more positions.        7. The polypeptide of any of paragraphs 1-6, which is a fragment        of SEQ ID NO: 30 or SEQ ID NO: 2, wherein the fragment has        trehalase activity.        8. The polypeptide of any of paragraphs 1-7, having at least        85%, at least 90%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 96%, at least 97%, at least        98%, at least 99% or 100% sequence identity to the mature        polypeptide of SEQ ID NO: 4.        9. The polypeptide of any of paragraphs 1-8, which is encoded by        a polynucleotide that hybridizes under very high stringency        conditions with (i) the mature polypeptide coding sequence of        SEQ ID NO: 3, (ii) the cDNA sequence thereof, or (iii) the        full-length complement of (i) or (ii).        10. The polypeptide of any of paragraphs 1-9, which is encoded        by a polynucleotide having at least 70%, at least 75%, at least        80%, at least 85%, at least 90%, at least 91%, at least 92%, at        least 93%, at least 94%, at least 95%, at least 96%, at least        97%, at least 98%, at least 99% or 100% sequence identity to the        mature polypeptide coding sequence of SEQ ID NO: 3 or the cDNA        sequence thereof.        11. The polypeptide of any of paragraphs 1-10, comprising or        consisting of SEQ ID NO: 4 or the mature polypeptide of SEQ ID        NO: 4 shown as amino acids 21-690 of SEQ ID NO: 4.        12. The polypeptide of any of paragraphs 1-11, which is a        variant of the mature polypeptide of SEQ ID NO: 4 comprising a        substitution, deletion, and/or insertion at one or more        positions.        13. The polypeptide of any of paragraphs 1-12, which is a        fragment of SEQ ID NO: 4 wherein the fragment has trehalase        activity.        14. A composition comprising the polypeptide of any of        paragraphs 1-13.        15. A whole broth formulation or cell culture composition        comprising the polypeptide of any of paragraphs 1-14.        16. A polynucleotide encoding the polypeptide of any of        paragraphs 1-14.        17. A nucleic acid construct or expression vector comprising the        polynucleotide of paragraph 16 operably linked to one or more        control sequences that direct the production of the polypeptide        in an expression host.        18. A recombinant host cell comprising the polynucleotide of        paragraph 16 operably linked to one or more control sequences        that direct the production of the polypeptide.        19. A method of producing the polypeptide of any of paragraphs        1-14, comprising cultivating a cell, which in its wild-type form        produces the polypeptide, under conditions conducive for        production of the polypeptide.        20. The method of paragraph 19, further comprising recovering        the polypeptide.        21. A method of producing a polypeptide having trehalase        activity, comprising cultivating the host cell of paragraph 18        under conditions conducive for production of the polypeptide.        22. The method of paragraph 21, further comprising recovering        the polypeptide.        23. A transgenic plant, plant part or plant cell transformed        with a polynucleotide encoding the polypeptide of any of        paragraphs 1-14.        24. A method of producing a polypeptide having trehalase        activity, comprising cultivating the transgenic plant or plant        cell of paragraph 21 under conditions conducive for production        of the polypeptide.        25. The method of paragraph 24, further comprising recovering        the polypeptide.        26. A process of producing a fermentation product, comprising    -   (a) liquefying a starch-containing material with an        alpha-amylase; optionally pre-saccharifying the liquefied        material before step (b);    -   (b) saccharifying the liquefied material;    -   (c) fermenting using a fermentation organism;    -   wherein        -   i) a glucoamylase;        -   ii) a trehalase of any of paragraphs 1-13;        -   iii) optionally a cellulolytic enzyme composition and/or a            protease;            are present and/or added during    -   saccharification step (b);    -   fermentation step (c);    -   simultaneous saccharification and fermentation;    -   optionally presaccharification step before step (b).        27. The process of paragraph 26, wherein the alpha-amylase is a        bacterial alpha-amylase, in particular of the genus Bacillus,        such as a strain of Bacillus stearothermophilus, in particular a        variant of a Bacillus stearothermophilus alpha-amylase, such as        the one shown in SEQ ID NO: 3 in WO 99/019467 or SEQ ID NO: 18        herein.        28. The process of paragraph 27, wherein the Bacillus        stearothermophilus alpha-amylase or variant thereof is        truncated, preferably to be from 485-495 amino acids long, such        as around 491 amino acids long.        29. The process of any of paragraphs 26 or 28, wherein the        Bacillus stearothermophilus alpha-amylase has a double deletion        at positions 1181+G182, and optionally a N193F substitution, or        deletion of R179+G180 (using SEQ ID NO: 18 for numbering).        30. The process of any of paragraphs 26-29, wherein the Bacillus        stearothermophilus alpha-amylase has a substitution in position        S242, preferably a S242A, E or Q substitution (using SEQ ID NO:        18 for numbering).        31. The process of any of paragraphs 26-30, wherein the Bacillus        stearothermophilus alpha-amylase has a substitution in position        E188, preferably E188P substitution (using SEQ ID NO: 18 for        numbering).        32. The process of any of paragraphs 26-31, wherein the        alpha-amylase in liquefaction step (a) is selected from the        following group of Bacillus stearothermophilus alpha-amylase        variants:    -   I181*+G182*+N193F+E129V+K177L+R179E;    -   I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S    -   I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+Q254S+M284V;    -   I181*+G182*+N193F+V59A+E129V+K177L+R179E+Q254S+M284V and    -   I181*+G182*+N193F+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S        (using SEQ ID NO: 18 herein for numbering).        33. The process of any of paragraphs 26-32, wherein the        glucoamylase is of fungal origin, preferably from a strain of        Aspergillus, preferably A. niger, A. awamori, or A. oryzae; or a        strain of Trichoderma, preferably T. reesei; or a strain of        Talaromyces, preferably T. emersonii, or a strain of        Gloeophyllum, such as G. serpiarium or G. trabeum.        34. The process of any of paragraphs 26-33, wherein the        glucoamylase is derived from Talaromyces emersonii, such as the        one shown in SEQ ID NO: 13 herein.        35. The process of any of paragraphs 26-34, wherein the        glucoamylase is selected from the group consisting of:        (i) a glucoamylase comprising the mature polypeptide of SEQ ID        NO: 13 herein;        (ii) a glucoamylase comprising an amino acid sequence having at        least 60%, at least 70%, e.g., at least 75%, at least 80%, at        least 85%, at least 90%, at least 91%, at least 92%, at least        93%, at least 94%, at least 95%, at least 96%, at least 97%, at        least 98%, or at least 99% identity to the mature polypeptide of        SEQ ID NO: 13 herein.        36. The process of any of paragraphs 26-35, wherein the        glucoamylase is derived from Gloeophyllum serpiarium, such as        the one shown in SEQ ID NO: 19 herein.        37. The process of any of paragraphs 26-36, wherein the        glucoamylase is selected from the group consisting of:        (i) a glucoamylase comprising the mature polypeptide of SEQ ID        NO: 19 herein;        (ii) a glucoamylase comprising an amino acid sequence having at        least 60%, at least 70%, e.g., at least 75%, at least 80%, at        least 85%, at least 90%, at least 91%, at least 92%, at least        93%, at least 94%, at least 95%, at least 96%, at least 97%, at        least 98%, or at least 99% identity to the mature polypeptide of        SEQ ID NO: 19 herein.        38. The process of any of paragraphs 26-37, wherein the        glucoamylase is derived from Gloeophyllum trabeum such as the        one shown in SEQ ID NO: 20 herein.        39. The process of any of paragraphs 26-38, wherein the        glucoamylase present and/or added in saccharification is        selected from the group consisting of:        (i) a glucoamylase comprising the mature polypeptide of SEQ ID        NO: 20 herein;        (ii) a glucoamylase comprising an amino acid sequence having at        least 60%, at least 70%, e.g., at least 75%, at least 80%, at        least 85%, at least 90%, at least 91%, at least 92%, at least        93%, at least 94%, at least 95%, at least 96%, at least 97%, at        least 98%, or at least 99% identity to the mature polypeptide of        SEQ ID NO: 20 herein.        40. The process of any of paragraphs 26-39, further wherein an        alpha-amylase is present and/or added during saccharification        step (b); fermentation step (c); simultaneous saccharification        and fermentation; or the optional presaccharification step        before step (b).        41. The process of paragraph 40, wherein the alpha-amylase is of        fungal or bacterial origin.        42. The process of paragraph 40 or 41, wherein the alpha-amylase        is derived from a strain of the genus Rhizomucor, preferably a        strain the Rhizomucor pusillus, such as the one shown in SEQ ID        NO: 3 in WO 2013/006756, such as a Rhizomucor pusillus        alpha-amylase hybrid having an Aspergillus niger linker and        starch-bonding domain, such as the one shown in SEQ ID NO: 15        herein.        43. The process of any of paragraphs 40-42, wherein the        alpha-amylase present and/or added in saccharification and/or        fermentation is selected from the group consisting of:        (i) an alpha-amylase comprising the mature polypeptide of SEQ ID        NO: 15 herein;        (ii) an alpha-amylase comprising an amino acid sequence having        at least 60%, at least 70%, e.g., at least 75%, at least 80%, at        least 85%, at least 90%, at least 91%, at least 92%, at least        93%, at least 94%, at least 95%, at least 96%, at least 97%, at        least 98%, or at least 99% identity to the mature polypeptide of        SEQ ID NO: 15 herein.        44. The process of any of paragraphs 40-43, wherein the        alpha-amylase is a variant of the alpha-amylase shown in SEQ ID        NO: 15 having at least one of the following substitutions or        combinations of substitutions: D165M; Y141W; Y141R; K136F;        K192R; P224A; P224R; S123H+Y141W; G20S+Y141W; A76G+Y141W;        G128D+Y141W; G128D+D143N; P219C+Y141W; N142D+D143N; Y141W+K192R;        Y141W+D143N; Y141W+N383R; Y141W+P219C+A265C; Y141W+N142D+D143N;        Y141W+K192R V410A; G128D+Y141W+D143N; Y141W+D143N+P219C;        Y141W+D143N+K192R; G128D+D143N+K192R; Y141W+D143N+K192R+P219C;        G128D+Y141W+D143N+K192R; or G128D+Y141W+D143N+K192R+P219C (using        SEQ ID NO: 15 for numbering).        45. The process of any of paragraphs 36-44, wherein the        alpha-amylase is derived from a Rhizomucor pusillus, in        particular with an Aspergillus niger glucoamylase linker and        starch-binding domain (SBD), preferably the one disclosed as SEQ        ID NO: 15 herein, preferably having one or more of the following        substitutions: G128D, D143N, preferably G128D+D143N (using SEQ        ID NO: 15 for numbering).        46. The process of any of paragraphs 44-45, wherein the        alpha-amylase variant has at least 60% identity, such as at        least 70%, preferably at least 75% identity, preferably at least        80%, more preferably at least 85%, more preferably at least 90%,        more preferably at least 91%, more preferably at least 92%, even        more preferably at least 93%, most preferably at least 94%, and        even most preferably at least 95%, such as even at least 96%, at        least 97%, at least 98%, at least 99%, but less than 100%        identity to the mature part of the polypeptide of SEQ ID NO: 15        herein.        47. The process of any of paragraphs 26-46, wherein the        cellulolytic enzyme composition is derived from Trichoderma        reesei, Humicola insolens or Chrysosporium lucknowense.        49. The process of any of paragraphs 26-48, wherein the        cellulolytic enzyme composition comprising a beta-glucosidase, a        cellobiohydrolase, an endoglucanase and optionally a GH61        polypeptide.        50. The process of any of paragraph 26-49, wherein the        cellulolytic enzyme composition comprises a beta-glucosidase,        preferably one derived from a strain of the genus Aspergillus,        such as Aspergillus oryzae, such as the one disclosed in WO        2002/095014 or the fusion protein having beta-glucosidase        activity disclosed in WO 2008/057637, or Aspergillus fumigatus,        such as one disclosed in SEQ ID NO: 2 in WO 2005/047499 or SEQ        ID NO: 10 herein or an Aspergillus fumigatus beta-glucosidase        variant disclosed in WO 2012/044915; in particular an        Aspergillus fumigatus beta-glucosidase variant with one or more,        such as all, of the following substitutions: F100D, S283G,        N456E, F512Y; or a strain of the genus a strain Penicillium,        such as a strain of the Penicillium brasilianum disclosed in WO        2007/019442, or a strain of the genus Trichoderma, such as a        strain of Trichoderma reesei.        51. The process of any of paragraphs 26-50, wherein the        cellulolytic enzyme composition comprises a cellobiohydrolase I        (CBH I), such as one derived from a strain of the genus        Aspergillus, such as a strain of Aspergillus fumigatus, such as        the Cel7a CBH I disclosed in SEQ ID NO: 6 in WO 2011/057140 or        SEQ ID NO: 6 herein, or a strain of the genus Trichoderma, such        as a strain of Trichoderma reesei.        52. The process of any of paragraphs 26-50, wherein the        cellulolytic enzyme composition comprises a cellobiohydrolase II        (CBH II, such as one derived from a strain of the genus        Aspergillus, such as a strain of Aspergillus fumigatus; such as        the one disclosed as SEQ ID NO: 8 herein or a strain of the        genus Trichoderma, such as Trichoderma reesei, or a strain of        the genus Thielavia, such as a strain of Thielavia terrestris,        such as cellobiohydrolase II CEL6A from Thielavia terrestris.        53. The process of any of paragraphs 26-52, wherein the        cellulolytic enzyme composition further comprises a GH61        polypeptide having cellulolytic enhancing activity such as one        derived from the genus Thermoascus, such as a strain of        Thermoascus aurantiacus, such as the one described in WO        2005/074656 as SEQ ID NO: 2 or SEQ ID NO: 21 herein; or one        derived from the genus Thielavia, such as a strain of Thielavia        terrestris, such as the one described in WO 2005/074647 as SEQ        ID NO: 7 and SEQ ID NO: 8; or one derived from a strain of        Aspergillus, such as a strain of Aspergillus fumigatus, such as        the one described in WO 2010/138754 as SEQ ID NO: 1 and SEQ ID        NO: 2; or one derived from a strain derived from Penicillium,        such as a strain of Penicillium emersonii, such as the one        disclosed in WO 2011/041397 as SEQ ID NO: 2 or SEQ ID NO: 12        herein.        54. The process of any of paragraphs 26-54, wherein the        cellulolytic enzyme composition is a Trichoderma reesei        cellulolytic enzyme composition, further comprising Thermoascus        aurantiacus GH61A polypeptide having cellulolytic enhancing        activity (SEQ ID NO: 2 in WO 2005/074656 or SEQ ID NO: 21        herein) and Aspergillus fumigatus beta-glucosidase (SEQ ID NO: 2        of WO 2005/047499) or SEQ ID NO: 6 herein.        55. The process of any of paragraphs 26-54, wherein the        cellulolytic enzyme composition is a Trichoderma reesei        cellulolytic enzyme composition further comprising Penicillium        emersonii GH61A polypeptide having cellulolytic enhancing        activity disclosed in WO 2011/041397 as SEQ ID NO: 2 or SEQ ID        NO: 12 herein; and Aspergillus fumigatus beta-glucosidase (SEQ        ID NO: 2 of WO 2005/047499 or SEQ ID NO: 10 herein) or a variant        thereof with one or more, such as all, of the following        substitutions: F100D, S283G, N456E, F512Y.        56. The process of any of paragraphs 26-55, wherein the        cellulolytic enzyme composition is dosed from 0.0001-3 mg EP/g        DS, preferably 0.0005-2 mg EP/g DS, preferably 0.001-1 mg/g DS,        more preferred from 0.005-0.5 mg EP/g DS, even more preferred        0.01-0.1 mg EP/g DS.        57. The process of any of paragraphs 26-56, wherein the        presaccharification is carried out at a temperature from 40-75°        C., such as 50-70° C., preferably 60° C.; a pH between 4-6,        preferably 5; for a period of 30-360 minutes, such as from        60-420 minutes, such as around between 150-180 minutes.        58. A process of any of paragraphs 26-57, comprising the steps        of:    -   (a) liquefying a starch-containing material with an        alpha-amylase;    -   optionally pre-saccharifying the liquefied material before step        (b);    -   (b) saccharifying the liquefied material;    -   (c) fermenting using a fermentation organism;    -   wherein        -   i) a glucoamylase;        -   ii) a trehalase of any of paragraphs 1-13;            are present and/or added during    -   saccharification step (b);    -   fermentation step (c);    -   simultaneous saccharification and fermentation;    -   optionally presaccharification step before step (b).        59. A process of any of paragraphs 26-58, comprising the steps        of:    -   (a) liquefying a starch-containing material with an        alpha-amylase;    -   optionally pre-saccharifying the liquefied material before step        (b);    -   (b) saccharifying the liquefied material;    -   (c) fermenting using a fermentation organism;    -   wherein        -   i) a glucoamylase from Talaromyces emersonii or Gloeophyllum            serpiarium;        -   ii) a trehalase shown in any of paragraphs 1-13;            are present and/or added during    -   saccharification step (b);    -   fermentation step (c);    -   simultaneous saccharification and fermentation;    -   optionally presaccharification step before step (b).        60. A process of any of paragraphs 26-58, comprising the steps        of:    -   (a) liquefying a starch-containing material with an        alpha-amylase;    -   optionally pre-saccharifying the liquefied material before step        (b);    -   (b) saccharifying the liquefied material;    -   (c) fermenting using a fermentation organism;    -   wherein        -   i) a glucoamylase from Talaromyces emersonii or Gloeophyllum            serpiarium;        -   ii) a trehalase shown any of paragraphs 1-13;        -   iii) a cellulolytic enzyme composition derived from            Trichoderma reesei;            are present and/or added during    -   saccharification step (b);    -   fermentation step (c);    -   simultaneous saccharification and fermentation;    -   optionally presaccharification step before step (b).        61. The process of any of paragraphs 26-60, wherein        saccharification step (a) and fermentation step (b) are done        separately or simultaneously.        62. The process of any of paragraphs 26-61, wherein the        fermentation product is recovered after fermentation.        63. The process of any of paragraphs 26-62, wherein the        starch-containing material is plant material selected from the        corn (maize), cobs, wheat, barley, rye, milo, sago, cassava,        tapioca, sorghum, rice, peas, beans, sweet potatoes, or a        mixture thereof, preferably corn.        64. The process of any of paragraphs 26-63, wherein the        temperature in liquefaction is above the initial gelatinization        temperature, in particular in the range from 70-100° C., such as        between 75-95° C., such as between 75-90° C., preferably between        80-90° C., such as 82-88° C., such as around 85° C.        65. The process of any of paragraphs 26-64, wherein liquefaction        step (a) is carried out at a pH in the range between 3 and 7,        preferably from 4 to 6, or more preferably from 4.5 to 5.5.        66. The process of any of paragraphs 26-65, wherein the dry        solid content (DS) in liquefaction lies in the range from 20-55        wt.-%, preferably 25-45 wt.-%, more preferably 30-40 wt.-% or        30-45 wt-%.        66. The process of any of paragraphs 26-65, further comprises,        prior to the liquefaction step (a), the steps of:

x) reducing the particle size of the starch-containing material,preferably by dry milling;

y) forming a slurry comprising the starch-containing material and water.

67. The process of any of paragraphs 26-66, wherein a jet-cooking stepis carried out prior to liquefaction in step (a).

68. The process of any of paragraphs 26-67, wherein thestarch-containing material is reduced in particle size, such by drymilling or wet milling or using particle size emulsion technology.

69. The process of any of paragraphs 26-68, wherein the fermentation iscarried out for 30 to 150 hours, preferably 48 to 96 hours.

70. The process of any of paragraphs 26-69, wherein the temperatureduring fermentation in step (b) or simultaneous saccharification andfermentation in steps (a) and (b) is between 25° C. and 40° C.,preferably between 28° C. and 36° C., such as between 28° C. and 35° C.,such as between 28° C. and 34° C., such as around 32° C.71. The process of any of paragraphs 26-70, wherein further a proteaseis present during saccharification and/or fermentation.72. The process of any of paragraphs 26-71, wherein glucoamylase ispresent and/or added in an amount of 0.001 to 10 AGU/g DS, preferablyfrom 0.01 to 5 AGU/g DS, especially 0.1 to 0.5 AGU/g DS.73. The process of any of paragraphs 26-72, wherein the fermentationproduct is an alcohol, preferably ethanol, especially fuel ethanol,potable ethanol and/or industrial ethanol.74. The process of any of paragraphs 26-73, further wherein a proteaseis present and/or added during

-   -   saccharification step (b);    -   fermentation step (c);    -   simultaneous saccharification and fermentation;    -   optionally presaccharification step before step (b).        75. The process of any of paragraphs 26-74, wherein the protease        is derived from Thermoascus, in particular Thermoascus        aurantiacus, especially Thermoascus aurantiacus CGMCC No. 0670        (classified as EC 3.4.24.39) shown in SEQ ID NO: 17 herein.        76. The process of paragraph 75, wherein the protease is the one        shown in SEQ ID NO: 17 herein or a protease being at least 60%,        such as at least 70%, such as at least 80%, such as at least        90%, such as at least 95%, such as at least 96%, such as at        least 97%, as as at least 98%, such as at least 99% identical to        SEQ ID NO: 17 herein.        77. The process of any of paragraphs 26-74, wherein the protease        is derived from a strain of Meripilus, in particular Meripilus        giganteus, in particular the one shown as SEQ ID NO: 32 herein.        78. The process of paragraph 77, wherein the protease is the one        shown in SEQ ID NO: 32 herein or a protease being at least 60%,        such as at least 70%, such as at least 80%, such as at least        90%, such as at least 95%, such as at least 96%, such as at        least 97%, as as at least 98%, such as at least 99% identical to        SEQ ID NO: 32 herein.        79. The process of any of paragraphs 26-78, wherein the        fermenting organism is derived from a strain of Saccharomyces,        such as Saccharomyces cerevisae.        80. A process of producing fermentation products from        starch-containing material comprising:    -   (i) saccharifying a starch-containing material at a temperature        below the initial gelatinization temperature; and    -   (ii) fermenting using a fermentation organism;    -   wherein saccharification and/or fermentation is done in the        presence of the following enzymes: glucoamylase, alpha-amylase,        trehalase of any of paragraphs 1-13, and optionally a protease        and/or a cellulolytic enzyme composition.        81. A process of producing a fermentation product from        pretreated cellulosic material, comprising:    -   (a) hydrolyzing said pretreated cellulosic material with a        cellulolytic enzyme composition;    -   (b) fermenting using a fermenting organism; and    -   (c) optionally recovering the fermentation product,    -   wherein a trehalase of any of paragraphs 1-13 is added and/or        present in hydrolysis step (a) and/or fermentation step (b).        82. The process of any of paragraphs 26-81, wherein the        trehalase is added in an amount between 0.01-20 ug EP        trehalase/g DS, such as between 0.05-15 ug EP terhalase/g DS,        such as between 0.5 and 10 ug EP trehalase/g DS.        83. The process of any of paragraphs 81-82, wherein the        cellulolytic enzyme composition is derived from Trichoderma        reesei, Humicola insolens or Chrysosporium lucknowense.        84. The process of any of paragraphs 81-83, wherein the        cellulolytic enzyme composition comprising a beta-glucosidase, a        cellobiohydrolase, an endoglucanase and optionally a GH61        polypeptide.        85. The process of any of paragraph 81-84, wherein the        cellulolytic enzyme composition comprises a beta-glucosidase,        preferably one derived from a strain of the genus Aspergillus,        such as Aspergillus oryzae, such as the one disclosed in WO        2002/095014 or the fusion protein having beta-glucosidase        activity disclosed in WO 2008/057637, or Aspergillus fumigatus,        such as one disclosed in SEQ ID NO: 2 in WO 2005/047499 or SEQ        ID NO: 10 herein or an Aspergillus fumigatus beta-glucosidase        variant disclosed in WO 2012/044915; in particular an        Aspergillus fumigatus beta-glucosidase variant with one or more,        such as all, of the following substitutions: F100D, S283G,        N456E, F512Y; or a strain of the genus a strain Penicillium,        such as a strain of the Penicillium brasilianum disclosed in WO        2007/019442, or a strain of the genus Trichoderma, such as a        strain of Trichoderma reesei.        86. The process of any of paragraphs 81-85, wherein the        cellulolytic enzyme composition comprises a cellobiohydrolase I        (CBH I), such as one derived from a strain of the genus        Aspergillus, such as a strain of Aspergillus fumigatus, such as        the Cel7a CBH I disclosed in SEQ ID NO: 6 in WO 2011/057140 or        SEQ ID NO: 6 herein, or a strain of the genus Trichoderma, such        as a strain of Trichoderma reesei.        87. The process of any of paragraphs 81-86, wherein the        cellulolytic enzyme composition comprises a cellobiohydrolase II        (CBH II, such as one derived from a strain of the genus        Aspergillus, such as a strain of Aspergillus fumigatus; such as        the one disclosed as SEQ ID NO: 8 herein or a strain of the        genus Trichoderma, such as Trichoderma reesei, or a strain of        the genus Thielavia, such as a strain of Thielavia terrestris,        such as cellobiohydrolase II CEL6A from Thielavia terrestris.        88. The process of any of paragraphs 81-87, wherein the        cellulolytic enzyme composition further comprises a GH61        polypeptide having cellulolytic enhancing activity such as one        derived from the genus Thermoascus, such as a strain of        Thermoascus aurantiacus, such as the one described in WO        2005/074656 as SEQ ID NO: 2 or SEQ ID NO: 21 herein; or one        derived from the genus Thielavia, such as a strain of Thielavia        terrestris, such as the one described in WO 2005/074647 as SEQ        ID NO: 7 and SEQ ID NO: 8; or one derived from a strain of        Aspergillus, such as a strain of Aspergillus fumigatus, such as        the one described in WO 2010/138754 as SEQ ID NO: 1 and SEQ ID        NO: 2; or one derived from a strain derived from Penicillium,        such as a strain of Penicillium emersonii, such as the one        disclosed in WO 2011/041397 as SEQ ID NO: 2 or SEQ ID NO: 12        herein.        89. The process of any of paragraphs 81-88, wherein the        cellulolytic enzyme composition is a Trichoderma reesei        cellulolytic enzyme composition, further comprising Thermoascus        aurantiacus GH61A polypeptide having cellulolytic enhancing        activity (SEQ ID NO: 2 in WO 2005/074656 or SEQ ID NO: 21        herein) and Aspergillus fumigatus beta-glucosidase (SEQ ID NO: 2        of WO 2005/047499) or SEQ ID NO: 6 herein.        90. The process of any of paragraphs 81-89, wherein the        cellulolytic enzyme composition is a Trichoderma reesei        cellulolytic enzyme composition further comprising Penicillium        emersonii GH61A polypeptide having cellulolytic enhancing        activity disclosed in WO 2011/041397 as SEQ ID NO: 2 or SEQ ID        NO: 12 herein; and Aspergillus fumigatus beta-glucosidase (SEQ        ID NO: 2 of WO 2005/047499 or SEQ ID NO: 10 herein) or a variant        thereof with one or more, such as all, of the following        substitutions: F100D, S283G, N456E, F512Y.        91. The process of any of paragraphs 81-90, wherein the        cellulolytic enzyme composition is dosed from 0.0001-3 mg EP/g        DS, preferably 0.0005-2 mg EP/g DS, preferably 0.001-1 mg/g DS,        more preferred from 0.005-0.5 mg EP/g DS, even more preferred        0.01-0.1 mg EP/g DS.

The invention claimed is:
 1. A process of producing a fermentationproduct, comprising steps: (a) liquefying a starch-containing materialwith an bacterial alpha-amylase; (b) saccharifying the starch-containingliquefied material; and (c) fermenting the starch-containing liquefiedmaterial from step (b) using yeast as a fermentation organism in afermentation reaction, wherein the fermentation reaction comprises anenzymatic composition which comprises: i) a fungal glucoamylase, and ii)a fungal alpha amylase, and a polypeptide having trehalase activity,wherein the polypeptide having trehalase activity is having at least 95%amino acid sequence identity to the amino acid sequence of the maturepolypeptide of SEQ ID NO: 30, and wherein said mature polypeptide of SEQID NO: 30 consists the amino acid sequence from position 21 to position694 of the amino acid sequence as set forth in SEQ ID NO:
 30. 2. Theprocess of producing a fermentation product according to claim 1,wherein the process comprises pre-saccharifying the starch-containingliquefied material before step (b).
 3. The process of producing afermentation product according to claim 1, wherein said enzymaticcomposition is present or added during said saccharification step (b),or said fermentation step (c).
 4. The process of producing afermentation product according to claim 2, wherein said enzymaticcomposition is present or added during said pre-saccharification.
 5. Theprocess of producing a fermentation product according to claim 1,wherein said mature polypeptide of SEQ ID NO: 30 is encoded by apolynucleotide having at least 90% nucleotide sequence identity to themature polypeptide SEQ ID NO: 30 coding nucleotide sequence as set forthin SEQ ID NO:
 29. 6. The process of claim 1, wherein the polypeptidehaving trehalase activity has the amino acid sequence of the maturepolypeptide of SEQ ID NO:
 30. 7. The process of claim 1, wherein thefermentation reaction further comprises a serine protease or metalloprotease.
 8. The process of claim 1, wherein the fermentation reactionfurther comprises a cellulolytic composition comprising fungalcellulases.
 9. The process of claim 8, wherein the cellulolyticcomposition comprises a Penicillium emersonii GH61A polypeptide.
 10. Theprocess of claim 8, wherein the cellulolytic composition comprises anAspergillus fumigatus beta-glucosidase.
 11. The process of claim 8,wherein the cellulolytic composition comprises an Aspergillus fumigatuscellobiohydrolase.
 12. The process of claim 11, wherein the Aspergillusfumigatus cellobiohydrolase is a cellobiohydrolase I (CBHI).
 13. Theprocess of claim 11, wherein the Aspergillus fumigatus cellobiohydrolaseis a cellobiohydrolase II (CBHII).
 14. The process of claim 8, whereinthe cellulolytic composition comprises an Aspergillus fumigatuscellobiohydrolase I (CBHI) and an Aspergillus fumigatuscellobiohydrolase II (CBHII).